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HEMATOPOIESIS
From the Department of Cell Biology and Anatomy,
Medical University of South Carolina, Charleston, SC.
WNT proteins compose a family of secreted signaling molecules that
regulate cell fate and behavior. The possible influence of WNTs on
hematopoietic cell fate was examined. Both hematopoietic progenitor cell (HPC)-enriched embryonic avian bone marrow cells and
the quail mesodermal stem cell line QCE6 were used for these studies.
Under optimized conditions, the bone marrow and QCE6 cells behaved
identically and developed into red blood cells (RBCs), monocytes,
macrophages, granulocytes, and thrombocytes. This broad range of blood
cell phenotypes exhibited by QCE6 cells was dependent on their active
expression of WNT11. However, when QCE6 cells were prevented from
producing WNT11 The various cell types that make up the blood are
thought to emanate from a common multipotent stem cell of mesodermal
origin. During embryogenesis, blood cell formation results from at
least 2 independent episodes. Hematopoietic cells are first observed within the blood islands of early somite stage embryos. These cellular
aggregates arise from ventral mesodermal cells that have migrated to
extra-embryonic regions during gastrulation. These "primitive"
hematopoietic progenitor cells (HPCs) will yield primarily erythrocytes, which provide circulating blood cells for the developing embryo. Shortly thereafter, intra-embryonic dorsal mesoderm contributes to a second wave of blood cell formation Although avian hematopoiesis is very similar to mammalian
hematopoiesis, there are several noticeable differences. First, red
blood cells (RBCs) are nucleated.5 Second,
heterophils, which along with basophils and eosinophils compose the
granulocytic cell types in the bird, are not exhibited in mammals.
Third, thrombocytes, which are equivalent to the nonnucleated mammalian
platelet, are the progeny of mononuclear thromboblasts.6
In comparison with mouse and human studies, the field of avian
hematopoiesis has been hindered by the absence of suitable cell
identification markers. Very few clusters-of-differentiation antibodies
exist that react to avian leukocytes.7 Hence, cytological
examination with azure stains (eg, May-Grünwald-Giemsa and
Wright-Giesma) has remained the predominant means of identifying
specific blood cell types in the bird. Additionally, the detection of
leukocyte-associated enzymatic activities, such as acid phosphatase
(AP), tartaric acid-resistant phosphatase (TARP), naphthol AS-D
chloroacetate esterase (CAE), and A large number of individual extracellular signaling factors that
regulate hematopoietic cell differentiation have been described. Many
of these factors In prior investigations, we examined the ability of WNTs to promote the
differentiation of mesoderm during early
embryogenesis.18,19 To assist in these studies, we used
the mesodermal stem cell line QCE6 as a cell culture model for studying
the regulated diversification of mesoderm. QCE6 cells were derived from
the mesodermal layer of early gastrula stage Japanese quail
(Coturnix coturnix japonica) embryos20,21 and
possess the potential to differentiate into cardiomyocytes, endothelial
cells, and RBCs.22 For the present study, we examined
whether QCE6 cells have the capability to give rise to other blood
lineages in addition to erythrocytes. Here, we report that QCE6 cells
will exhibit as diverse a hematopoietic potential as HPCs taken
directly from quail bone marrow. Since the ability of QCE6 cells to
undergo cardiomyocyte differentiation was WNT dependent,18
we additionally asked whether WNT expression would influence the blood
cell differentiation of these cells. We found that WNTs profoundly
affect the diversity of hematopoietic cell phenotypes that are
exhibited by both QCE6 and bone marrow-derived cells. Specifically, we
find that WNT signals inhibit the formation of macrophages
and increase the prevalence of monocyte and erythrocyte cell phenotypes.
Isolation of avian bone marrow cells
Methylcellulose cultures
Fibrin gel cultures Purified avian bone marrow or QCE6 cells were plated at 5 × 104 cells per milliliter culture in medium containing DMEM, 1 mg/mL human fibrinogen (Sigma), 20% fetal bovine serum, 10% chick serum (heat inactivated for 1 hour at 56°C), 5% WeHi-3-conditioned medium, 1% deionized bovine serum albumin, 1 IU/mL erythropoietin, 100 ng/mL SCF, 500 ng/mL hydrocortisone, 27.4 mmol/L NaHCO3, 200 µg/mL iron-saturated transferrin, and pen/strep. The mixture was allowed to clot at 37°C for 15 minutes following the addition of 1 IU/mL bovine thrombin (Sigma). Gels were overlaid with an equal volume of medium without fibrinogen to inhibit dehydration. Cultures were plated in duplicate and incubated for up to 7 days at 37°C in a 5% CO2 atmosphere.Stably transfected QCE6 cell sublines The production of stable transfectants of QCE6 cells with altered levels of WNT11 expression has been previously described.18 Briefly, full-length WNT11 complementary DNA (cDNA), in either sense or antisense orientations, was inserted immediately downstream of the cytomegalovirus promoter in the eukaryotic expression vector pcDNA3 (Invitrogen, Carlsbad, CA) and then introduced into QCE6 cells by means of LipofectAMINE (Gibco BRL). Cells that stably incorporated the transgenes were selected by their resistance to neomycin and subsequently cloned. Multiple subclones that contained either the sense or the antisense WNT11 cDNA were generated and referred to as WNT11ox (ox stands for overexpress) and WNT11 s cells, respectively. For the present study, we
exclusively used a single WNT11 sense clone, WNT11ox/3, and 2 individual antisense subclones, WNT11s/4 and WNT11s/6. To
maintain expression of the transgenes, these stably transfected cells
were continuously passaged in the presence of 200 µg/mL G418 (Gibco
BRL). To determine relative levels of WNT11 protein produced by
WNT11ox/3, QCE6, WNT11s/6, and WNT11s/4 cells were grown in 60-mm
tissue-culture dishes until confluency was reached. The medium was
removed from each dish and replaced with 5 mL minimal essential medium
(MEM) containing 5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL selenium (Sigma) plus 50 µg/mL heparin (which solubilizes WNT
protein bound to cell surfaces and matrix). Following overnight
incubation, the heparin-treated media were collected and assayed for
WNT11 protein expression by immunoblot analysis as
described.19 To ensure that the volumes of secreted
protein used for blotting represented equal numbers of cells, total
cell protein was prepared from these cultures following collection of
the culture fluids. Culture plates were scraped after the addition of
hypotonic buffer (5 mmol/L Tris pH 7.5, 5 mmol/L egtazic acid, 5 mmol/L
EDTA, 2 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL Leupeptin, 1 µg/mL Pepstatin A); the resulting cell lysates were passed several
times through 25-gauge needles, incubated on ice for 2 hours, and
brought to isotonicity with 10 × phosphate-buffered saline (PBS).
These protein samples were electrophoretically separated on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis gels, transferred
to polyvinylidene fluoride (PVDF) membrane, and probed with
streptavidin-conjugated alkaline phosphatase (Tropix, Bedford, MA) to
detect the mitochondrial protein pyruvate
carboxylase.27,28
For producing WNT5a-expressing QCE6 cells, we followed a similar
protocol except that the expression vector employed was
pcDNA3.1/myc-6xHis A (Invitrogen). This allowed for the
production of a WNT5a fusion protein with a myc-6xHis tag at
the carboxy terminus. To accomplish this, Xenopus WNT5a cDNA
(a gift from Randall Moon) was amplified by polymerase chain reaction
(PCR) to produce full coding length sequence that included 31 base
pairs of the 5'-untranslated region up to the last amino-acid codon.
The PCR fragment, which contained terminal EcoRI and XbaI sites from
the PCR primers, was subsequently ligated in-frame with
myc-6xHis tag of pcDNA3.1A. Following LipofectAMINE-mediated transfection and G418 selection, individual clones were isolated by
means of cloning rings. After expansion, culture medium was collected
from these clones and immunoblotted for myc-tagged fusion protein Generation of WNT-containing conditioned media and purification of WNT5a protein Conditioned media were harvested from confluent cultures of either WNT11ox/3 or WNT5a/QCE6 cells grown in serum-free media containing 5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL selenium, and 0.4% albumin in vitamin-enriched MEM (Sigma). The presence of WNT11 and WNT5a protein within these conditioned media was verified by immunoblotting by means of WNT11-specific18,19 or myc-tag-reactive (Santa Cruz Biotechnology) antibodies, respectively. Purification of WNT5a protein from conditioned media was accomplished by means of nickel-nitrilotriacetic acid (Ni-NTA) spin columns (Qiagen Inc, Valencia, CA), which binds 6xHis-tagged proteins with high affinity. Conditioned media from WNT5a/QCE6 cells (WNT5a CM) was supplemented with imidazole (Qiagen) at a final concentration of 5 mmol/L. Individual Ni-NTA columns were pre-equilibrated with NaH2PO4 pH8, 300 mmol/L NaCl, and 5 mmol/L imidazole and then loaded with 500 µL aliquots of WNT5a CM per 5 mmol/L imidazole. After samples were passed through the columns by low-speed centrifugation (at 700g), columns were washed 4 times with NaH2PO4 pH8, 300 mmol/L NaCl, and 20 mmol/L imidazole. Metal-binding protein was collected by eluting twice from each column with 200 µL volumes of NaH2PO4 pH8, 300 mmol/L NaCl, and 250 mmol/L imidazole. Afterward, pooled samples were dialyzed 4 times against 500 volumes of PBS and stored at 70°C. Protein concentrations of WNT5a-containing
conditioned media and Ni-NTA column-purified WNT5a-containing
fraction were determined by means of the Micro BCA protein assay
reagent kit (Pierce, Rockford, IL), according to the manufacturer's instructions.
To assess the influence of ectopic WNT protein on blood cell diversification, embryonic bone marrow cells, QCE6 cells, or QCE6 stably transfectants were plated in either methylcellulose (MeC) or fibrin gel (FB) cultures as described above, except that conditioned medium collected from either WNT11ox/3 or WNT5a/QCE6 cells was substituted for DMEM. Alternatively, Ni-NTA column-purified WNT5a protein was added to the cultures at a final concentration of 5% vol/vol. Assays for cell differentiation Both MeC and FB cultures were monitored microscopically each day for cell growth and differentiation. Cultures were scored in situ according to number and cell phenotype of colonies, with medium (20 to 50 cells) and large (more than 50 cells) colonies tabulated independently. Prior to staining, live cultures were documented with an Olympus IMT-2 inverted microscope with the use of Ektachrome ASA 100 film (Kodak, Rochester, NY). Both MeC and FB cultures were processed for May-Grünwald-Giemsa,6 esterase, and phosphatase stains. For MeC cultures, similar colonies were picked and cytospun onto electrostatically charged microscope slides (Fisher Scientific, Suwanee, GA). For FB cultures, gels were transferred onto microscope slides and partially dehydrated according to Lanotte's technique.29 Designation of blood cell phenotypes within these cultures, following May-Grünwald-Giemsa staining, was according to previous documentation of avian blood cells.6 Naphthol AS-D CAE, ANAE, AP, and TARP stains were performed by means of Sigma kits 90-A1, 90-C2, and 387-A.
Hematopoietic potential of avian embryonic bone marrow To examine the molecular regulation of HPC diversification, we first optimized conditions for eliciting a full range of blood cell phenotypes from embryonic avian bone marrow. Hematopoietic cell progenitors were obtained from the bone marrow by separation of cell suspensions on Ficoll-Paque gradients and subsequent removal of adherent cells by overnight incubation on tissue-culture plastic (see "Materials and methods"). Staining of bone marrow cells before (Figure 1A) or after (Figure 1B) Ficoll-Paque purification with May-Grünwald-Giemsa (MGG) solution verified that these purification procedures removed most differentiated cells and left primarily nondifferentiated cells having a blast-like cell morphology (Figure 1B).
Extensive experimentation led to the development of culture conditions
for promoting complete hematopoiesis in both MeC suspension cultures
and fibrin (FB) gels (or plasma clot gels), the latter being the most
common method used for avian hematopoiesis.26,30-32 Similar to our previous experience with culturing avian mesodermal cells,22 we found that a mixture of chicken and bovine
sera, in addition to cytokines, provided optimal culture conditions for
supporting cell growth and differentiation. Under these conditions, in
both MeC and FB gels, 6 morphologically distinct colonies were observed: erythroid (E) (Figure 1C), granulocyte/macrophage (GM) (Figure 1D), granulocyte/erythroid/macrophage (GEM) (Figure 1E), granulocyte (G), macrophage/erythroid (ME) and macrophage (M) (Figure
1F). Quantitation of the MeC cultures (Figure
2) showed that the most frequently
occurring colony types were erythroid and granulocyte/macrophage, which
yielded 134 and 181 total colonies per 5 × 104 bone
marrow cells. Macrophage colonies also occurred at a high rate;
however, most of the macrophage-only clusters were of smaller (20 to 50 cell) size. The bone marrow culture data shown in this report used ED14
quail embryos as the tissue source. Additionally, these culture
conditions were replicated for analogously staged ED16 chick bone
marrow, which produced identical results.
Avian bone marrow cells cultured in either MeC or FB gels displayed
maximal colony growth and cell differentiation by day 6 of incubation.
At this time, cell phenotype was analyzed further by harvesting the
cultures and staining them with MGG solution. Cytological examination
indicated that bone marrow-derived cells were able to produce colonies
containing RBCs, granulocytes, monocytes, macrophages and
thrombocytes
Hematopoietic potential of QCE6 cells Having ascertained optimal protocols for embryonic avian bone marrow hematopoiesis, we next examined the diversification of the mesodermal stem cell line QCE6 under the same conditions. As reported previously,22 QCE6 cells have the capacity to differentiate to RBCs. To investigate their full hematopoietic potential, we plated QCE6 cells in conditions devised for bone marrow-derived HPCs. As was observed with avian embryonic HPCs (Figure 3), QCE6 cells cultured in either MeC or FB gels gave rise to a broad range of blood cell phenotypes (Figure 4). Specifically, as indicated by the pattern of MGG staining, QCE6 cells differentiated into RBCs, granulocytes, monocytes, macrophages, and thrombocytes (Figure 4).
As an additional means to compare the cell phenotypes that developed
from bone marrow and QCE6 cells, we examined MeC and FB gel cultures
for the distribution of specific enzyme activities associated with
leukocytes: AP, TARP, naphthol AS-D CAE, and ANAE. The results we
obtained with avian bone marrow cells were consistent with those
reported by other investigators.35 All leukocytes that
developed from avian bone marrow exhibited positive AP activity (Figure
5A). Addition of tartaric acid limited
this AP activity (ie, TARP) to macrophage subpopulations. Both CAE and
ANAE activity were detected in granulocytes, macrophages, and
monocytes. Similarly to mouse and human, the ANAE enzyme activity was
fluoride sensitive in monocytes8 and therefore was greatly
reduced in these cells following NaF treatment (Figure 5B). These
patterns of enzyme activity appeared to be similarly exhibited by QCE6
cells. For example, all leukocytes that developed from QCE6 cells
exhibited AP activity (Figure 5C), with TARP activity restricted to
macrophages (Figure 5D). Also, as with bone marrow-derived cells, QCE6
cells exhibiting granulocyte, macrophage, or monocyte phenotypes were both CAE-positive (Figure 5E) and ANAE-positive. Again, as observed with bone marrow-derived cells, monocytes were the only QCE6-derived cell type whose ANAE activity was inhibited by treatment with NaF
(Figure 5F). Together, these data indicate that QCE6 cells will exhibit
a differentiation potential similar to bone marrow HPCs when placed in
a culture environment that promotes hematopoiesis.
WNT11 expression alters hematopoietic diversification of QCE6 cells The findings on the hematopoietic potential of QCE6 cells suggest that they may have utility as a cell line model for examining the regulated diversification of blood cell progenitors. In prior studies,18,19 we examined the function of WNT-secreted signaling proteins in diverting mesodermal progenitors to cardiac phenotypes. Since blood cell progenitors have been shown to respond to WNT signals,16,17 we investigated whether WNTs may similarly influence hematopoietic cell diversification. As an initial inquiry into this possibility, we examined the blood cell potential of QCE6 cells as a function of WNT expression. QCE6 cells express WNT11 messenger RNA and protein, a characteristic they share with a subset of nondifferentiated mesodermal cells in the early gastrula.18 Previously, we described the generation of QCE6 subclones with altered levels of WNT11 expression.18 This was accomplished by stably transfecting QCE6 cells with transgenes that expressed either WNT11 sense or antisense RNA, yielding variant subclones that produce either constitutively higher or diminished levels of WNT11 protein, respectively. For the present study, we have used an individual WNT11 sense QCE6 stable transfectant cell line, referred to as WNT11ox/3, and 2 individually derived WNT11 antisense sublines of QCE6 cells: WNT11s/4 and WNT11s/6. The relative levels of WNT11 produced by these cell lines were assayed by
immunoblot analyses (Figure 6).
In parallel with the parental QCE6 cells, these various QCE6 stably
transfected sublines were cultured in conditions that support
hematopoiesis (Figure 7). In common with
nontransfected QCE6 cells, WNTox/3 cells plated in MeC or FB gels
produced a broad range of blood cell phenotypes, which included RBCs,
granulocytes, monocytes, and thrombocytes. However, WNT11ox/3 cells
displayed a shift toward the erythrocyte lineage, with an increase in
both the frequency of erythrocyte-containing colonies (Figure 7) and the proportion of erythroblasts (Figure
8A) and mature RBCs (Figure 8B-C) within
these colonies. Another feature of WNT11ox/3 hematopoiesis was a
dramatic decrease in macrophage formation, with a corresponding increase in the numbers of cells exhibiting a monocyte or promonocyte morphology (Figure 7). In contrast, highly vacuolated macrophages dominated the MeC and FB gel cultures of WNT11
As a further test on the ability of WNTs to control blood cell
diversification, we stably introduced into QCE6 cells a transgene encoding for WNT5a. We chose WNT5a for these studies since its functional properties closely resemble those of
WNT11,36,37 and it is expressed within both embryonic and
adult hematopoietic tissue.16,17 To facilitate detection
of WNT5a protein by QCE6 stably transfected sublines, this molecule was
expressed as a fusion protein with a myc-6xHis tag at the
carboxy end. Accordingly, expression of WNT5a protein was demonstrated
by immunoblotting for the myc epitope tag (Figure
9A). As was observed for the
overexpression of WNT11 (Figure 8A-C), cells that expressed WNT5a
showed a phenotypic shift toward the RBC lineage (Figure 8F), with
macrophage formation occurring very infrequently (Figure 7).
Exposure to WNT protein shifts the blood cell phenotypes displayed
by WNT11 s cells
exhibit a shift in cell fate toward macrophage formation. To verify
that the inhibition of WNT11 production is principally responsible for
the cell demographic profile exhibited by WNT11s cells, we investigated the response of these cells to exogenously added WNT11 or
WNT5a. In a previous study, we demonstrated that conditioned media from
WNT11ox cells contains functionally active WNT11
protein.19 Similarly, WNT5a/QCE6 cells produce high
amounts of soluble secreted WNT5a protein (Figure 9B). Thus,
WNT11s/4 cells were plated in FB gels in the absence or presence of
either WNT11ox- or WNT5a/QCE6-conditioned media (WNT11 CM or WNT5a CM).
In contrast to the outcome in the absence of WNT-containing media
(Figure 7), WNT11s cells treated with either WNT11 CM or WNT5a CM
generated significant numbers of RBCs. Also, the proportion of
monocytes was increased in response to these treatments. To verify that
this phenotypic shift was in fact due to WNT protein, we purified WNT5a
from conditioned media. Taking advantage of the carboxy-terminal 6xHis
fusion tag, we separated WNT5a protein from contaminating proteins on
the basis of its affinity for Ni+ (Figure 9B). As observed
with the conditioned media treatments, purified WNT5a protein provoked
WNT11s cells to give rise predominantly to monocytes (Figure
10A) and RBCs (Figure 10B).
Exposure to WNT protein shifts the blood cell phenotypes displayed by bone marrow-derived HPCs The above results demonstrated that WNTs are able to regulate the diversification of blood phenotypes exhibited by QCE6 cells. To extend these findings, we went back to studying blood cell progenitors obtained from avian bone marrow. Quail HPCs were plated in FB gels, in the presence or absence of conditioned media containing WNT11 or WNT5a. WNT11 CM (Figure 10C-D) and WNT5a CM treatment yielded similar outcomes, with cultures containing monocytes (Figure 10C), RBCs (Figure 10D), granulocytes, and thrombocytes. More importantly, in comparison with the control cultures, macrophage formation was markedly reduced. Instead, the conditioned media greatly increased the occurrence of monocytes (and to a lesser extent RBCs) as compared with cells cultured in the absence of ectopic WNT protein (Figure 11). When we repeated these experiments with quail bone marrow cells using purified WNT5a protein, the same result was produced: greater numbers of monocytes (Figure 10E) and RBCs (Figure 10F), with macrophages appearing very infrequently in these cultures (Figure 11).
Previous investigations have shown that WNTs may act as
determiners of cell lineage,14,15 either by pushing
nondifferentiated cells toward more specialized
phenotypes,38,39 shifting cells between alternative cell
fates,40,41 or both. Here, we report that WNT proteins can
significantly influence the phenotypic distribution of blood
cells QCE6 cells serve as a cell line model of hematopoiesis The QCE6 cell line was derived from anterior mesodermal cells of an early gastrula stage quail embryo,20,21 analogous to Hamburger and Hamilton42 stage 4. Although QCE6 cells were initially characterized for their cardiac and endothelial cell potential, it was subsequently demonstrated that these cells can develop into RBCs.22 Intrigued by this observation, we began to examine whether QCE6 cells might have an even broader cell potential. To investigate the capability of this cell line to give rise to nonerythroid blood cells, we first optimized culture conditions for blood cell diversification of hematopoietic progenitor cells. The HPC source used for these experiments was bone marrow from late-stage embryonic quail or chick embryos. Under optimized conditions, bone marrow-derived cells were able to develop into RBCs, granulocytes, monocytes, macrophages, and thrombocytes. When QCE6 cells were then cultured under the identical conditions, they also gave rise to the same broad range of blood cell types. The phenotypes of these cells were verified by cytological examination with May-Grünwald-Giemsa stain and the pattern of leukocyte-associated enzyme reactivity. Side-by-side comparison with bone marrow-derived cells clearly indicated that the cell types generated from QCE6 cells are differentiated RBCs, granulocytes, monocytes, macrophages, and thrombocytes.The differentiation of QCE6 cells into multiple hematopoietic cell types raises the issue of why these cells display this phenotypic potential. Although QCE6 cells were obtained from a mesodermal area that does not normally give rise to blood cells, other studies have shown that this region is capable of producing RBCs when cultured in the presence of yolk sac endoderm.43,44 The potential of early embryonic mesoderm to give rise to additional blood cell types has not been shown. Whether the multipotentiality of QCE6 cells, as evidenced by the data presented here, is totally reflective of the tissue from which it was derived is an issue for a later study. What is shown is that QCE6 cells will behave in a manner nearly identical to bone marrow HPCs when exposed to hematopoietic-promoting conditions. Thus, this cell line has utility as a tool to examine mechanisms that regulate hematopoiesis. This was dramatically illustrated when we examined the influence of WNT expression on the phenotypic distribution of blood cells, as information obtained from QCE6 cells appeared to be directly applicable for the regulated diversification of bone marrow HPCs. WNTs influence the diversification of blood cell progenitors QCE6 cells express WNT11 a property they share with a subset of
anterior mesodermal cells from the early gastrula.18 Under culture conditions optimized for hematopoiesis of bone marrow HPCs,
QCE6 cells will give rise to a similar broad range of differentiated blood cell types. However, alteration of WNT expression by these cells
greatly influenced the formation of specific blood cell phenotypes by
these cells. Inhibition of WNT11 production by QCE6 cells, via stable
transfection of a WNT11 antisense transgene, produced cultures that
were dominated by macrophages, produced few if any monocytes, and were
devoid of RBCs. Accordingly, the formation of RBCs and monocytes by
these WNT11s cells was restored when the cells were exposed to
ectopic WNT proteineither in the form of WNT11- or WNT5a-containing
conditioned media or as purified WNT5a protein. Moreover, when WNT
levels were increased in QCE6 cells by stable insertion of WNT11 or
WNT5a transgenes, the prevalence of RBCs and monocytes was
significantly increased, with the appearance of macrophages markedly
reduced in these cultures. When bone marrow HPCs were exposed to WNT11
or WNT5a, macrophages appeared very infrequently within the cultures.
Instead, monocytes were now the prevalent blood cell that arose from
the bone marrow-derived cells, with increased numbers of RBCs
also apparent.
The expression of WNT5a in the yolk sac and by hematopoietic
progenitors in the bone marrow16,17 implicates this
molecule as a candidate regulator of blood cell formation in situ. That WNT5a and WNT11 similarly influenced the distribution of blood cell
phenotypes in our cultures is consistent with previous observations that these 2 proteins share functional activities.36,37
Although WNTs are a highly conserved group of molecules, they do
exhibit dramatic differences in their functional activities. Thus, it has been useful to subdivide WNTs into 2 broad classes Diversification of blood cells In the culture conditions used for our experiments, both bone marrow HPCs and QCE6 cells gave rise to RBCs, granulocytes, monocytes, macrophages, and thrombocytes. When QCE6 cells were prevented from producing WNT11 (ie, WNT11s cells), they no longer generated RBCs,
and the appearance of monocytes was infrequent. Instead, cultures of
WNT11s cells were dominated by macrophages. Accordingly, WNT11s
cells behaved like normal QCE6 cells when ectopic WNT11 or WNT5a was
added to the cultures. Overexpressing WNT protein in QCE6 cells, with
either WNT11 or WNT5a transgenes, inhibited macrophage differentiation,
but increased the prevalence of RBCs and monocytes. Exposure of bone
marrow HPCs to either WNT11 or WNT5a similarly inhibited macrophage
formation, as macrophages were infrequently observed in these latter
cultures. In this instance, RBC numbers were moderately enhanced, with
a much greater enhancement of monocytes produced from the bone marrow
cells in response to the WNT signal. This relative enhancement by WNTs
on RBC and monocyte development was the principal difference we
observed in our results with bone marrow and QCE6 cells. This variance
may reflect that QCE6 cells, being a cloned cell line, present a less
complex cellular environment than does the relatively heterogeneous
population of bone marrow HPCs. An additional consideration is that
expression of WNT5a (or other WNTs) by quail bone marrow, as has been
shown for human bone marrow,17 may moderate the relative
enhancement of RBC formation by ectopically added WNT protein. Overall,
the QCE6 cells behaved remarkably similar to the bone marrow cells in
the MeC and FB gel cultures. Not only did this cell line generate the
same range of blood cell phenotypes as did bone marrow-derived hematopoietic progenitors, but also that macrophage development was
identically affected in response to altered WNT expression.
Together, the data derived from the bone marrow and QCE6 cells suggest that WNT5a or a similar signal (ie, WNT11) influences blood cell formation by shifting cell fate from a macrophage to an RBC phenotype. Interestingly, the same WNT signals also inhibit the differentiation of monocytes to macrophages. Thus, both WNT11 and WNT5a promote the expression of RBCs and monocytes at the expense of macrophages. These results raise the question of whether similar WNT responsiveness indicates linkage between 2 regulatory events that both have an impact on macrophage lineage. In other words, are RBCs and monocytes more closely linked than previously suspected?
We thank Randall Moon for his generosity in providing us with the Xenopus WNT5a cDNA.
Submitted January 5, 2000; accepted August 17, 2000.
Supported by National Institutes of Health grant HL55923 (C.A.E.), American Heart Association Grant 9808097U (C.A.E.), American Heart Association Grant-in-Aid 9950638N (L.M.E.), and South Carolina Research Initiative Grant (C.A.E., L.M.E.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Carol A. Eisenberg, Department of Cell Biology and Anatomy, Medical University of South Carolina, 173 Ashley Ave, Suite 652, PO Box 250508, Charleston, SC 29425; e-mail: eisenbec{at}musc.edu.
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S. K. Cho, A. Bourdeau, M. Letarte, and J. C. Zuniga-Pflucker Expression and function of CD105 during the onset of hematopoiesis from Flk1+ precursors Blood, December 15, 2001; 98(13): 3635 - 3642. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, BMP4, and stem
cell factor (SCF).
progenitor
cells in human bone marrow.
), 271 colonies for WNT11
), 405 colonies for WNT11ox/3
(
), and 401 colonies for WNT5a-transfected QCE6 (WNT5a/QCE6, 
).
The phenotypic groups indicated on the x-axis are colonies that
contained RBCs (Ery), macrophages plus monocytes (Mac + Mono),
monocytes without macrophages (Mono), and granulocytes (Gr). Note that
WNT11
