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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1608-1616
Mice Lacking Transcription Factor NF-E2 Provide In Vivo
Validation of the Proplatelet Model of Thrombocytopoiesis and Show
a Platelet Production Defect That Is Intrinsic to Megakaryocytes
By
Patrick Lecine,
Jean-Luc Villeval,
Paresh Vyas,
Bethany Swencki,
Yuhui Xu, and
Ramesh A. Shivdasani
From the Department of Adult Oncology, Dana-Farber Cancer Institute,
Boston, MA; the Department of Medicine, Children's Hospital Medical
Center, Boston, MA; and the Department of Medicine, Harvard Medical
School, Boston, MA.
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ABSTRACT |
Mechanisms of platelet production and release by mammalian
megakaryocytes are poorly understood. We used thrombocytopenic knockout
mice to better understand these processes. Proplatelets are filamentous
extensions of terminally differentiated megakaryocytes that are thought
to represent one mechanism of platelet release; however, these
structures have largely been recognized in cultured cells and there has
been no correlation between thrombocytopoiesis in vivo and proplatelet
formation. Mice lacking transcription factor NF-E2 have a late arrest
in megakaryocyte maturation, resulting in profound thrombocytopenia. In
contrast to normal megakaryocytes, which generate abundant
proplatelets, cells from these mice never produce proplatelets, even
after prolonged stimulation with c-Mpl ligand. Similarly,
megakaryocytes from thrombocytopenic mice with lineage-selective loss
of transcription factor GATA-1 produce proplatelets very rarely. These
findings establish a significant correlation between thrombocytopoiesis
and proplatelet formation and suggest that the latter represents a
physiologic mechanism of platelet release. We further show that
proplatelet formation by normal megakaryocytes and its absence in cells
lacking NF-E2 are independent of interactions with adherent (stromal)
cells. Similarly, thrombocytopenia in NF-E2 / mice
reflects intrinsic defects in the megakaryocyte lineage. These
observations improve our understanding of platelet production and
validate the study of proplatelets in probing the underlying mechanisms.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CIRCULATING BLOOD platelets develop
within the cytoplasm of megakaryocytes, the rarest of mammalian
hematopoietic cells. In the course of their differentiation,
megakaryocytes acquire several unique attributes, including polyploid
DNA content, platelet-specific granules, and an elaborate system of
demarcation membranes. Mature megakaryocytes finally release thousands
of platelets by mechanisms that are poorly understood and
controversial. Whereas aspects of lineage specification, endomitosis,
and cytoplasmic maturation have been studied through a combination of
cell culture, hematopoietic colony assays, and electron microscopy,
these approaches have led only to inferences about the dynamic process
by which megakaryocytes ultimately release platelets. The extreme
rarity of megakaryocytes in sites of hematopoiesis limits
identification of cells in the act of releasing platelets.
Two nonmutually exclusive mechanisms of platelet release have been
proposed. According to one model, terminally mature megakaryocytes leave their site of origin intact or nearly intact and are fragmented within the first capillary bed they encounter in the systemic circulation, usually the lungs.1 Observations of a
difference between the number of megakaryocyte fragments found in the
venous and arterial circulation2,3 and, rarely, of entire
megakaryocytes in blood vessels4,5 are consistent with this
model. The alternative model proposes that the bulk of
thrombocytopoiesis occurs at the site of megakaryocyte maturation. To
provide a mechanism for platelet release in situ, investigators have
pointed out that mature megakaryocytes can extend long cytoplasmic
processes, designated as proplatelets6,7 or compound
platelets,8 that are comprised of nascent blood platelets
in a tandem array. However, criticisms of the phenomenon of proplatelet
generation include the fact that that it has largely been recognized in
vitro and frequently only after nonphysiologic manipulations.9-12 More importantly, this model lacks in
vivo correlation with platelet production per se.
Studies in thrombocytopoiesis have been facilitated greatly by the
identification of the major cytokine that regulates megakaryocyte growth and differentiation, the ligand for the c-Mpl
receptor.13-15 The c-Mpl ligand is an
erythropoietin-related glycoprotein that greatly increases platelet
counts as a single agent in mice and humans; a wealth of data now
points to the c-Mpl ligand as a regulator of all aspects of
megakaryocyte differentiation, including endomitosis and cytoplasmic
maturation (reviewed in Kaushansky16). Although mature
human megakaryocytes cultured in the presence of the c-Mpl ligand
clearly generate proplatelets,17,18 a genuine correlation between proplatelet formation in vitro and thrombocytopoiesis in vivo
has remained elusive.
We recently reported the phenotype of knockout mice lacking the
hematopoietic-specific basic-leucine zipper (bZip) transcription factor
p45 NF-E2.19 The most dramatic aspect of these animals is a
profound thrombocytopenia, resulting from an arrest in late megakaryocyte cytoplasmic maturation. The megakaryocytes of p45 NF-E2/ mice show a large cytoplasm with
abundant demarcation membranes but few platelet-specific granules.
Subsequently, we generated mice with megakaryocyte-selective loss of
expression of the zinc-finger transcription factor
GATA-120; these mice also are severely thrombocytopenic as
a result of arrested megakaryocyte differentiation. Despite extensive
characterization of megakaryocyte morphology in these mutant mice, the
cellular and molecular basis of thrombocytopenia are as obscure as the mechanisms of platelet formation and release by normal megakaryocytes.
We report here on our studies on three aspects of thrombocytopoiesis by
normal and mutant megakaryocytes. First, megakaryocytes cultured from
murine fetal livers in the presence of the c-Mpl ligand develop large
numbers of proplatelets. In contrast, megakaryocytes lacking NF-E2
never generate proplatelets and GATA-1-deficient megakaryocytes do so
very rarely. These findings provide a strong correlation between
proplatelet formation in vitro and thrombocytopoiesis in vivo and lend
support to the view that proplatelet formation represents a physiologic
mechanism of platelet release. Second, we provide more detailed
characterization of normal murine proplatelets. Finally, we demonstrate
that proplatelet formation by normal megakaryocytes in vitro and
thrombocytopenia resulting from absence of NF-E2 in vivo occur
independently of interactions with other cell types. The sum of these
observations furthers our understanding of thrombocytopoiesis and
enhances the utility of genetic models of thrombocytopenia to analyze
the mechanism of platelet production.
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MATERIALS AND METHODS |
Megakaryocyte culture.
p45 NF-E2 and GATA-1 heterozygous mice were maintained on an inbred
129/Sv genetic background. Livers were recovered from mouse fetuses
between embryonic day (E) 13 and 15. Single-cell suspensions, prepared
by successive passage through 22- and 25-gauge needles, were cultured
in Dulbecco's modified Eagle's medium (GIBCO BRL, Bethesda, MD)
supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin, and 0.1 µg/mL polyethylene
glycol-conjugated recombinant human c-Mpl ligand (Amgen, Thousand Oaks,
CA). Between the days 4 and 6, these fetal liver cultures contained
50% to 60% acetylcholinesterase-positive cells. c-Mpl ligand was
added only at the initiation of liquid cultures.
Conditioned medium was obtained from megakaryocyte liquid cultures on
day 5 and contaminating cells were removed by passage through 0.45-µm
filters. Undiluted conditioned medium, without determination of
cytokine content, was used to replace the original medium in
experiments testing its role in influencing proplatelet formation.
To establish stromal cell layers, fetal liver or bone marrow cells were
cultured under identical conditions as described above and all
nonadherent cells were removed by multiple washes with phosphate-buffered saline on day 5 of culture. Fresh fetal liver suspensions, depleted of greater than 95% adherent cells by three successive incubations on tissue culture-treated plastic plates over 8 to 10 hours, were then cultured on the stromal cell layers, as
described above.
To generate megakaryocyte colony-forming units (CFU-Mk) in semisolid
medium, 1 to 5 × 105 fetal liver cells were cultured
in 1.2 mL of 50% methylcellulose in Iscove's modified Dulbecco
medium, supplemented with 30% fetal bovine serum (Stem Cell
Technologies, Vancouver, British Columbia, Canada) and 0.1 µg/mL
c-Mpl ligand, as described above. Proplatelet formation was studied
after 7 days in culture at 37°C.
Detection and characterization of proplatelets.
Proplatelets were detected and scored by phase contrast microscopy of
cells growing in suspension in liquid culture. A total of
103 to 105 cells were cytocentrifuged onto
coated glass slides and acetylcholinesterase activity was detected as
previously reported.21 For indirect immunofluorescence,
cytocentrifuged cell preparations were fixed in methanol for 1 minute,
washed, blocked with 2% goat serum in tris-buffered saline, and
successively incubated with 1:100 dilutions of rabbit antimouse
platelet antiserum (gift of C.W. Jackson, St Jude Children's Research
Hospital, Memphis, TN) and fluorescein isothiocyanate
(FITC)-conjugated goat antirabbit IgG (Pharmingen, Los
Angeles, CA).
Electron microscopy.
To preserve proplatelet integrity, cells were concentrated by gentle
centrifugation (200g for 4 minutes) and then adhered by gravity
to poly-L-lysine (Sigma, St Louis, MO) -coated glass cover slips
resting in a Petri dish. After 15 minutes, fixative (1.5%
glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.4) was added slowly
into the Petri dish and the cells were fixed for 4 hours. After
embedding in Epoxy resin in an inverted Beem capsule, the single layer
of megakaryocytes was detached from the cover slips by immersion in
liquid nitrogen, and ultrathin sections were cut with a Dupont MT6000
microtome (Dupont, Newton, CT), stained with uranyl acetate and lead
citrate, and examined with a JEOL 100CX-II transmission electron
microscope (JEOL, Peabody, MA) at an accelerating voltage of 60 kV.
For scanning electron microscopy (SEM), cover slips with cells were
taken through a series of alcohols and dried using a Ladd Model 28000 Critical Point Dryer (Ladd Research Industries, Inc, Hatfield, PA). The
cover slips were then mounted onto SEM specimen tubs, sputter-coated
with Polaron SEM coating system (Polaron Instruments, Inc, Burlington,
VT), and examined with a JEOL JSM-35CF Scanning Electron Microscope at
an accelerating voltage of 20 kV.
Fetal liver transplantation.
Livers were recovered from the fetuses of p45 NF-E2 heterozygote
intercrosses on postcoital day 14 and single-cell suspensions were
prepared by passage through a syringe and 22-gauge needle. Pending
determination of the p45 NF-E2 genotype of each fetus, these cells were
cultured overnight in Dulbecco's modified Eagle's medium supplemented
with 20% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL
streptomycin, and 10% spleen cell conditioned medium. The next day, 14 adult 129/SvJ adult females were treated with whole-body irradiation at
two doses of 500 cGy each 3 hours apart. Six mice were then injected
intravenously with 6 to 8 × 106 fetal liver cells
derived from p45 NF-E2+/+ or p45 NF-E2+/
donors and 6 mice with the same number of cells cultured from p45
NF-E2/ donor fetuses. One recipient from the
test group and 2 mice from the control group showed either endogenous
or chimeric reconstitution at 3 to 5 weeks, indicating sublethal
irradiation; the analysis presented here is limited to the majority of
mice with complete hematopoietic reconstitution by the donor cells.
Histologic analysis of reconstituted spleen and bone marrow and
Southern analysis, using a flanking genomic DNA probe, were
performed as described previously.19 At various times, 50 to 100 µL of blood was removed from the retro-orbital sinus and
diluted in Unopette buffer (Becton Dickinson, Franklin, NJ), and
leukocyte and platelet counts were determined by manual counting
under light microscopy.
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RESULTS |
Abundant proplatelet formation by cultured murine megakaryocytes.
We cultured murine fetal liver cells in liquid or semisolid medium in
the presence of fetal bovine serum and the c-Mpl ligand and observed
the cultures over several days. Under these conditions, mature
megakaryocytes develop in large numbers and produce an impressive array
of cell projections (Fig 1a) that appear
identical to structures previously described in human,17,22
rat,10 guinea pig,11 and bovine12
megakaryocytes cultured in vitro. These proplatelets, as they have been
termed,6 are seen very rarely when c-Mpl ligand is excluded
from the cultures. The formation of proplatelets correlates well with
the degree of megakaryocyte maturity, being undetectable over the first
2 days and then increasing in frequency as the culture becomes
dominated by large, polyploid, acetylcholinesterase-positive cells; in
wild-type or p45 NF-E2+/ megakaryocyte cultures,
40% to 50% of the acetylcholinesterase-positive cells display
proplatelets between culture days 4 and 6. In contrast, megakaryocytes
lacking NF-E2 grow appreciably larger than control cells and acquire
morphologic and molecular markers of maturity but are never observed to
generate proplatelets (Fig 1b), even after prolonged culture. This
finding provides the first correlation between proplatelet formation in
vitro and thrombocytopoiesis in vivo.
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| Fig 1.
Phase-contrast micrographs of control (a; +/+) and
NF-E2-deficient (b;  /) megakaryocytes on day 5 of culture of
fetal liver cells in the c-Mpl ligand. The hematopoietic cells growing
in suspension, and easily distinguished from the adherent stromal cells
(straight arrow in [a], right panel), are mostly large and small
megakaryocytes but include few myeloid and erythroid cells. Wild-type
cultures (a) show abundant proplatelets characterized by platelet-size
structures (curved arrows in [a]) on an extensive system of
filamentous processes. Mutant cultures (b), here shown at higher cell
density to include more cells in each microscopic field, never produce
proplatelets. Original magnification × 200.
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Proplatelet formation has not previously been reported in cells
isolated from the laboratory mouse, a species in which genetic models
of thrombocytopenia are available. In large part, this reflects the
relatively recent isolation of the c-Mpl ligand, which is required to
propagate and drive complete maturation of sufficient numbers of
megakaryocytes. A tendency to study cells derived from adult mice may
also have been a factor; in our hands, culture of bone marrow cells
under the same conditions as fetal liver cells yields significantly
fewer megakaryocytes. However, mature megakaryocytes cultured from
wild-type or p45 NF-E2+/ adult bone marrows also
produce large numbers of proplatelets over the same culture period as
do fetal liver cells, whereas cells derived from p45
NF-E2/ adults fail to do so (data not shown).
Biochemical characterization of proplatelets.
Although proplatelets are believed to form by eversion of the
megakaryocyte cytoplasm through the system of demarcation membranes, their specific composition is not fully characterized. Wild-type murine
proplatelets show strong acetylcholinesterase activity (Fig 2a), a specific marker of the rodent
megakaryocyte and platelet cytoplasm,21 throughout their
length. This finding indicates the incorporation of
cytoplasmic contents into these structures. Furthermore, a rabbit
antimouse platelet antiserum23 readily stains the surfaces
of the largest cells (megakaryocytes) in both control and p45
NF-E2/ cultures, including the full length of
proplatelets in the former (Fig 2b and c). Taken together, these data
suggest that proplatelet formation represents cytoplasmic and membrane
reorganization of mature megakaryocytes. This dramatic aspect of
terminal megakaryocyte differentiation is notably missing in cells
cultured from the profoundly thrombocytopenic mice lacking p45 NF-E2.
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| Fig 2.
Staining of cytocentrifuge preparations of wild-type (a
and b; +/+) and NF-E2-null (c; /) cultured megakaryocytes to
show cytoplasmic acetylcholinesterase activity (a) and immunoreactivity
with rabbit antimouse platelet antiserum (b and c). Proplatelets,
indicated by the large arrows in (a) and (b), are hence shown to
represent extensions of the megakaryocyte cytoplasm and membranes in
wild-type cells and are not detected in the mutant megakaryocytes.
Background staining of nonmegakaryocytic cells (short open bars) in (b)
and (c) and absence of cholinesterase staining in granulocytes and
monocytes (short straight arrows) in (a) provide internal controls for
the antiserum and enzyme staining, respectively. Original magnification × 200.
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Electron microscopy of proplatelets.
To better appreciate the structural basis of proplatelet formation in
wild-type megakaryocytes and its absence in cells lacking NF-E2, we
performed electron microscopy. Scanning electron micrographs of
megakaryocytes cultured from normal fetal livers provide
high-resolution images of the proplatelet surface and show these to be
structures that emanate from the cell (Fig
3a and b). The long filamentous connections between individual
platelet-size structures are also readily shown by this technique. In
contrast, the much larger megakaryocytes from NF-E2-deficient mice
(Fig 3c) show numerous small, villous projections but do not display
the proplatelets characteristic of normal mature megakaryocytes.
Transmission electron microscopy confirms that the proplatelet
cytoplasm is contiguous with that of the cell
(Fig 4). Although the static images
obtained by this technique are insufficient to draw conclusions about
the dynamic process by which proplatelets are generated by mature megakaryocytes, these data are consistent with internal fragmentation of the megakaryocyte cytoplasm, as suggested by earlier
studies.11,18,24 Indeed, ultrastructural features of
cytoplasmic reorganization that appear to precede proplatelet
formation, including dilatation of demarcation membranes, are also
notably absent from megakaryocytes lacking NF-E2 (data not shown).
Nevertheless, cultured p45 NF-E2/
megakaryocytes have many of the same properties seen in vivo, including
dearth of platelet-specific granules and abundance of demarcation
membranes, indicating that maturation of the cells in culture parallels
megakaryocyte differentiation in vivo.
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| Fig 3.
Scanning electron micrographs of p45
NF-E2+/+ (a), p45 NF-E2+/ (b), and p45
NF-E2/ (c) cultured megakaryocytes, showing extension
of proplatelets from within normal cells and the surface appearance of
the mutant cells, which are appreciably larger in size but do not
develop proplatelets. Note the long filamentous processes separating
individual platelet-size particles.
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| Fig 4.
Representative transmission electron micrograph of
cultured p45 NF-E2+/ megakaryocytes, showing the
proplatelet (arrowheads) as an extension of the cytoplasm.
Ultrastructural analysis of numerous p45 NF-E2/
megakaryocytes failed to show these structures.
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Impaired proplatelet formation by GATA-1-deficient megakaryocytes.
We have recently established a distinct murine model of
thrombocytopenia. Mice with a targeted deletion within the
5 -flanking region of the GATA-1 gene display
megakaryocyte-selective loss of GATA-1 expression, dysregulated growth
of megakaryocyte progenitors, and platelet counts that are reduced to
approximately 15% of normal.20 The ultrastructure of
GATA-1-deficient megakaryocytes shows a striking and early block in
cytoplasmic maturation, with features distinct from those observed in
the absence of p45 NF-E2. If proplatelet formation is a physiologically
important aspect of terminal megakaryocyte differentiation, then
GATA-1-deficient megakaryocytes also may be expected to be impaired in
their ability to generate these structures. Indeed, proplatelet
formation by GATA-1-deficient megakaryocytes is observed only rarely
in liquid (Fig 5a, see page 1611) or
semisolid (data not shown) cultures of the mutant cells or upon
staining of the mutant cells with acetylcholinesterase or antiplatelet
antiserum (Fig 5b and c, see page 1611). Scanning and transmission
electron microscopy (data not shown) confirm the virtual absence of
proplatelets and show cellular features similar to those observed in
megakaryocytes found in the spleen or bone marrow of mutant
animals,20 again providing correlation between our in vitro
and in vivo findings. Thus, the absence or rarity of proplatelet
formation by megakaryocytes cultured in the presence of the c-Mpl
ligand correlates with profound or moderately severe thrombocytopenia
in vivo in two distinct knockout mouse models of impaired megakaryocyte
differentiation.
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| Fig 5.
GATA-1-deficient megakaryocytes. Phase-contrast
micrograph (a), acetylcholinesterase staining (b), and indirect
immunofluorescence with a rabbit antimouse platelet antiserum (c) each
show the rarity of proplatelets among megakaryocytes cultured from
GATA-1-deficient mice. Control megakaryocytes (not shown here; see
Figs 1 and 2) displayed abundant proplatelets by each of these
techniques. For (a), (b), and (c), original magnifications × 200.
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Cell-autonomous lack of proplatelet formation.
Expression of p45 NF-E2 is restricted to hematopoietic tissues and
cultured cells of the erythroid, megakaryocyte, and mast cell
lineages.25 Although this expression pattern suggests that the thrombocytopenia seen in the absence of p45 NF-E2 results from a
requirement for NF-E2 function within the megakaryocyte itself, a role
for other cell types, particularly stromal cells, remains possible.
Indeed, previous studies have led to the hypothesis that various
aspects of megakaryocyte differentiation, including platelet release,
depend on interaction with the bone marrow stroma.26-28 Lack of proplatelet formation by p45 NF-E2/
megakaryocytes and the profound thrombocytopenia in NF-E2-null mice
provide opportunities to address this question. We refer here to
megakaryocytic processes that occur independently of other cells as
being cell-autonomous and any necessity for NF-E2 function within
megakaryocytes as reflecting a cell-autonomous requirement.
Supernatants from cultures of wild-type megakaryocytes fail to
stimulate proplatelet formation in p45 NF-E2/
cells; correspondingly, supernatants from the mutant cultures do not
inhibit proplatelet formation by wild-type cells
(Table 1). Previous studies have indicated
that mature human megakaryocytes can generate proplatelets even when
cultured as isolated cells in the absence of serum17,18;
however, these experiments were performed on late megakaryocyte progenitors (human CD34+ bone marrow cells further purified
by differential sedimentation or by flow cytometry for the CD38 marker)
and thus leave open the possibility that signals delivered at an
earlier stage in megakaryocyte differentiation are required for optimal
proplatelet production. We therefore depleted the adherent cell
fraction from a population of total wild-type fetal liver cells over
the first few hours after tissue harvest. Over the following week, the
remaining progenitors give rise to megakaryocytes that develop
proplatelets in similar numbers and at the same rate as cells from
nondepleted cultures (data not shown), indicating that contact with
stromal cells is not a requirement for proplatelet formation. Moreover, when p45 NF-E2/ fetal liver cells are
cocultured from the outset with adherent cells derived from normal
fetal livers or bone marrows, they remain unable to produce
proplatelets; similarly, coculture of wild-type fetal liver cells with
adherent cells of p45 NF-E2/ origin does not
inhibit proplatelet formation (Table 1). Hence, the lack of proplatelet
formation by the defective megakaryocytes is very likely a
cell-autonomous process reflecting a critical requirement for NF-E2
within the megakaryocyte lineage.
Cell-autonomous thrombocytopenia in the absence of NF-E2.
The mechanism underlying the inability of NF-E2-null megakaryocytes to
produce platelets is unclear. To determine whether this mechanism is
also cell-autonomous, we delivered lethal doses of  -irradiation to
wild-type adult mice and then attempted to rescue hematopoiesis in
these animals by introducing fetal liver cells derived from p45
NF-E2/ or control fetuses. All animals
reconstituted by wild-type donor cells recover normal hematologic
profiles, including platelet counts, within 3 weeks; in contrast, mice
reconstituted by NF-E2/ fetal liver cells
rapidly recover normal numbers of leukocytes (data not shown) but
remain profoundly thrombocytopenic  9 weeks after transplantation
(Fig 6a); complete reconstitution by cells of donor origin is indicated by Southern analysis of the hematopoietic tissues (Fig 6b). In recipients of p45 NF-E2-null fetal liver cells,
megakaryocytes harbor the same morphologic abnormalities seen in p45
NF-E2/ cells (Fig 6C). These findings
establish that the thrombocytopenia resulting from absence of NF-E2
function reflects deficiencies within the daughters of a
radiation-sensitive cell population, most likely the megakaryocyte
progenitors.
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| Fig 6.
Cell-autonomous thrombocytopenia in mice
lacking NF-E2 function. (a) Platelet counts from adult mice of the
indicated p45 NF-E2 genotype (left 2 bars, dark shading) are reproduced
after fetal liver cell transplantation. Lethally irradiated wild-type
recipients were transplanted with fetal liver cells from p45
NF-E2+/+ or NF-E2/ mice and
hematologic profiles followed for 3 to 9 weeks. Severe thrombocytopenia
is evident in recipients of p45 NF-E2/ transplants.
(b) Southern blot analysis of hematopoietic tissues from representative
fetal liver transplant recipients verifies reconstitution in these
animals by cells of the transplanted genotype. The wild-type allele is
represented by a 5-kb band, and the knockout allele is represented by a
7-kb band. (c) Hematoxylin and eosin-stained microscopic sections of
the spleen from representative recipient mice reconstituted with fetal
liver cells from wild-type (left panel) or p45 NF-E2/
(right 2 panels) donors. The numbers and morphology of the
megakaryocytes are similar to those seen in adults of the donor
genotypes. Original magnifications: for left two panels, ×40; for
right panel, ×100.
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DISCUSSION |
The cellular mechanisms by which megakaryocytes produce and release
millions of platelets daily are uncertain and difficult to study. In
large part, this is because megakaryocytes are among the rarest of
hematopoietic cells and because platelet release is a dynamic process
that occurs over an unspecified time period in an unknown location.
Nevertheless, ultrastructural analysis of megakaryocytes in vivo has
provided valuable insights into potential mechanisms of platelet
release; images of parasinusoidal megakaryocytes extending processes
that include nascent platelets (proplatelets) into the vascular
space7,26,29 led to the hypothesis that formation, and
subsequent fragmentation, of these structures contributes in some
manner to the pool of circulating platelets. Before the identification
of the c-Mpl ligand as the major regulator of megakaryocyte growth and
differentiation (reviewed in Kaushansky16), proplatelet
formation could consistently be demonstrated only in cells derived from
a limited number of species and under specific culture conditions
(reviewed in Choi30). Although wider use of the c-Mpl
ligand to culture megakaryocytes in vitro has led to increasing
recognition of the proplatelet as a genuine subcellular
entity,17,18 it has remained difficult to establish a link
between proplatelet formation in vitro and thrombocytopoiesis in vivo.
We show here that wild-type murine megakaryocytes develop a dramatic
array of proplatelets during culture in the c-Mpl ligand, thus
providing evidence that proplatelet formation is an integral aspect of
late megakaryocyte maturation in all species studied to date.
Furthermore, the kinetics of proplatelet formation closely parallels
megakaryocyte differentiation, ie, both the frequency of
proplatelet-bearing megakaryocytes and the average number of proplatelets per cell (data not shown) increase between 2 and 7 days of
culture, before cell viability decreases. These proplatelets express
both surface and cytoplasmic markers of the megakaryocyte/platelet lineage (Fig 2) and are shed into the culture medium in large numbers,
where they either appear as platelet-size particles or remain in the
beads on a string form that emanates from the mature megakaryocytes
(Figs 1 and 3).
Proplatelets are observed infrequently when either
human17,18 or murine (our observations) megakaryocytes are
cultured in the absence of the c-Mpl ligand. This may explain why these structures proved difficult to demonstrate in early studies of thrombocytopoiesis in mice and other species. Our demonstration of
abundant proplatelets in normal murine megakaryocytes may also be
traced in part to the choice of cultured tissue. Culture of adult bone
marrow or spleen cells under identical conditions yields many fewer
megakaryocytes than that of mouse fetal liver cells (data not shown),
perhaps reflecting the more limited proliferation potential of adult
progenitors. However, we do not observe substantial differences in the
acetylcholinesterase staining or ultrastructure of proplatelets
obtained from fetal and adult sources. Moreover, known features of
murine fetal liver-derived cells, such as the ability to produce
circulating platelets in the fetus and to reconstitute hematopoiesis in
lethally irradiated recipients, indicate that the fetal liver is a
physiologically relevant source of megakaryocytes for study.
Our most important observation is that proplatelets are absent from
cultures of p45 NF-E2/ megakaryocytes and
greatly reduced in cultures of GATA-1-deficient cells; the respective
knockout mice display either profound or moderately severe
thrombocytopenia. In each case, the development of megakaryocytes in
culture shows other features that are also seen in vivo, such as
unusually large megakaryocytes in the case of NF-E2 loss and smaller
megakaryocytes admixed with an excess of immature cells in the case of
GATA-1 deficiency (Vyas et al, manuscript in preparation).
The striking differences between the wild-type and mutant cells with
respect to generation of proplatelets thus establish a strong
correlation between proplatelet formation in vitro and platelet
production in vivo and support the view that proplatelets contribute to
the mechanism by which terminally differentiated megakaryocytes release
platelets.
Besides addressing the mechanism of platelet production in a species
that is amenable to genetic manipulation, our studies underscore the
value of examining this mechanism in culture, thus circumventing the
low likelihood of identifying platelet-producing megakaryocytes in
vivo. At the same time, it is worth emphasizing that the absence or
decrease of proplatelet formation by megakaryocytes lacking NF-E2 or
GATA-1, respectively, does not by itself point to the complete
mechanism of thrombocytopenia in these animal models. Although our
findings establish a critical link between proplatelet formation in
vitro and thrombocytopoiesis in vivo, the arrest in megakaryocyte
cytoplasmic maturation that characterizes these knockout
mice19,20 might well precede the maturational stage at
which generation of proplatelets is feasible. Although a requirement
for NF-E2 or GATA-1 in aspects of proplatelet production cannot be
ruled out, it is also possible that other cytoplasmic abnormalities of
the defective megakaryocytes, such as reduced numbers of granules and
disorganized demarcation membranes, simply preclude progression of the
cell through terminal differentiation, including proplatelet formation.
Early observations of proplatelet formation in vivo by megakaryocytes
in close contact with marrow stromal cells generated the notion that
platelet production might depend on such cell-cell interactions. We
have extended here previous studies17,18 to demonstrate
that proplatelet formation can occur independently of interactions
between megakaryocytes and stromal cells. The results summarized in
Table 1 further indicate that the lack of proplatelet formation in the
absence of NF-E2 function is not rescued by culture supernatant or
adherent (stromal) cells from wild-type cultures, suggesting that this
defect is intrinsic to the megakaryocyte. Additional proof for a
cell-autonomous defect in NF-E2-null megakaryocytes is provided by our
observation that the phenotype of NF-E2 knockout mice is reproduced in
its entirety in lethally irradiated wild-type mice that receive a
transplant of fetal liver cells derived from the knockout mice. Thus,
it is unnecessary to invoke the existence of a secreted
platelet-shedding factor solely on the basis of the phenotype of mice
lacking NF-E2. Although it is likely that megakaryocytes receive, and
depend on, signals delivered by other cells early in their
differentiation, our results with the normal and mutant cells argue
that terminal aspects of megakaryocyte differentiation, especially
proplatelet formation, are largely independent of such signals. One
might therefore speculate that platelet production and release are
programmed aspects of the maturing megakaryocyte once the cell has
committed to polyploidization and a megakaryocyte-specific pattern of
gene expression. Of course, this does not exclude the possibility that extraneous signals serve to modify the number or qualitative
characteristics of the proplatelets produced by mature megakaryocytes,
as suggested previously.9,31,32
In conclusion, we report that proplatelet formation is absent or
severely reduced in two distinct knockout mouse models of thrombocytopenia that result from primary disturbances in platelet production. These findings strongly suggest a physiologic role for
proplatelet formation in the normal mechanisms of platelet production
and release in vivo.
| |
FOOTNOTES |
Submitted February 24, 1998;
accepted May 6, 1998.
P.L. and J.-L.V. contributed equally to this study.
Supported by grants from the National Institutes of Health, the
Harcourt General Charitable Foundation, and the Dolphin Trust. P.L. was
supported by a fellowship from the Association pour la Recherche Contre
le Cancer (ARC, France), J.-L.V. by INSERM and the Fondation pour la
Recherche Medicale (France), and P.V. by a fellowship from the Wellcome
Foundation (UK).
Address reprint requests to Ramesh A. Shivdasani, MD, PhD, Dana-Farber
Cancer Institute, 44 Binney St, Boston, MA 02115; e-mail: ramesh_shivdasani{at}dfci.harvard.edu.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are grateful to Amgen, Inc for providing recombinant c-Mpl
ligand, to Carl Jackson for his generous gift of rabbit antimouse
platelet antiserum, and to Stuart Orkin for comments on the manuscript.
| |
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Abstract
Introduction
Abstract