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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02596
1 Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755,
USA.
2 Department of Anatomy, 1300 University Avenue, University of Wisconsin-Madison
School of Medicine and Public Health, Madison, WI 53706, USA.
Authors for correspondence (e-mail:
nancy.speck{at}dartmouth.edu;
kdowns{at}facstaff.wisc.edu)
Accepted 25 August 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Placenta, Allantois, Chorion, Hematopoiesis, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7.0
days postcoitum (dpc)] (Palis et al.,
1999
). The sites of hematopoietic cell emergence in mammals have
been defined using hematopoietic progenitor assays or, in the case of
hematopoietic stem cells, by transplantation into newborn, irradiated, or
otherwise compromised adult mice (for reviews, see
Dzierzak, 2005;
Speck et al., 2002). Sites
defined by these approaches include: the yolk sac, which gives rise to both
primitive and definitive blood cells; the aorta/gonad/mesonephros region and
its precursor, the para-aortic splanchnopleura (PAS); and the umbilical artery
(Cumano et al., 1996;
de Bruijn et al., 2000;
Medvinsky and Dzierzak, 1996;
Moore and Metcalf, 1970;
Müller et al., 1994;
Palis et al., 1999;
Weissman et al., 1978;
Yoder et al., 1997).
In addition to sites of hematopoietic cell emergence, two hematopoietic
cell niches have been identified within the liver and placenta in the
mid-gestation mouse conceptus (10.0-11.0 dpc). It has long been accepted
that the fetal liver is a site of hematopoietic colonization and is not an
intrinsic source of hematopoietic cells
(Houssaint, 1981). However, it
is not known whether the placenta, whose contribution to mammalian fetal
hematopoiesis was more recently appreciated
(Alvarez-Silva et al., 2003;
Gekas et al., 2005;
Melchers, 1979;
Ottersbach and Dzierzak, 2005)
is simply a site of colonization (like the fetal liver), or whether it is also
a source of hematopoietic cells. Determining whether any tissue is truly a
site of hematopoietic cell emergence is complicated by the fact that
hematopoietic cells circulate throughout the conceptus. Thus, in this study,
we have investigated the hematopoietic potential of the two individual
components of the murine placenta, the allantois and the chorion, prior to
establishment of vascular continuity within the conceptus.
The chorion arises during gastrulation (7.0-7.25 dpc) by displacement
of extra-embryonic ectoderm onto the roof of the newly formed exocoelomic
cavity (Snell and Stevens,
1966). It is a bilayered tissue of dual origin, composed of
extra-embryonic ectoderm derived from trophectoderm and extra-embryonic
mesoderm derived from proximal epiblast
(Gardner, 1983;
Kinder et al., 1999;
Lawson et al., 1991)
(Fig. 1A). The ectodermal
component of the chorion differentiates into syncytio- and cytotrophoblasts,
which mediate fetal/maternal exchange.
The murine allantois is thought to be composed wholly of extraembryonic
mesoderm (Duval, 1891) derived
from proximal epiblast that is transformed into mesoderm via passage through
the posterior primitive streak (Beddington,
1982; Copp et al.,
1986; Kinder et al.,
1999; Lawson et al.,
1991; Tam and Beddington,
1987). The allantois appears slightly later than the chorion
during gastrulation, and emerges first as a bud emanating from the posterior
region of the streak (7.25 dpc). The bud enlarges within the exocoelomic
cavity by a combination of mitosis, continued cellular addition from the
primitive streak and distal cavitation
(Brown and Papaioannou, 1993;
Downs and Bertler, 2000;
Ellington, 1985;
Tam and Beddington, 1987).
Ultimately, the allantois elongates far enough to make contact with the
chorionic mesoderm, and fuses with it [6-8 somite pairs (s), 8.5 dpc].
Chorio-allantoic union is mediated by the maturity of the allantois
(Downs and Gardner, 1995), and
requires both the cell-surface molecule vascular cell adhesion molecule 1
(VCAM1) on the outer mesothelial surface of the allantois
(Gurtner et al., 1995;
Kwee et al., 1995) and its
counter receptor 
4 integrin, on chorionic mesoderm
(Yang et al., 1995). Together
the allantois and chorion give rise to the labyrinth region of the
placenta.
Shortly after the allantoic bud is formed, allantoic mesoderm vascularizes
de novo (7.5 dpc). Vascularization occurs with distal-to-proximal
polarity, spreading toward the posterior end of the primitive streak
(Downs et al., 1998). At about
the same time, vasculogenesis takes place in the yolk sac blood islands and
heart region, and spreads toward the posterior streak. By 4-6 s, the three
major circulatory systems unite near the base of the allantois, forming a
vascular continuum throughout the conceptus
(Downs, 1998;
Inman and Downs, 2006). By 8 s
(8.5-8.75 dpc), primitive erythroid cells can be seen freely circulating
within the allantois (Downs et al.,
1998).
Although erythroid cells could be detected in the murine allantois in early
somite pair conceptuses, it was not entirely clear whether or not they
originated from the allantois. In avian embryos, the apparently
prevascularized allantois appears to be a hematopoietic tissue and contains
blood-island-like clusters of hematopoietic cells
(Caprioli et al., 1998;
Caprioli et al., 2001).
Dieterlen-Lièvre and colleagues also showed that the avian allantois
could contribute extensively to hematopoietic and endothelial cells in the
adult bone marrow following engraftment into the coelom of host embryos, and
thus has the potential to form definitive (adult) blood
(Caprioli et al., 1998). In
contrast, when headfold-stage murine allantoises were labeled intrinsically
with lacZ and extrinsically with tritiated thymidine, and then grafted into
the exocoelomic cavity of unlabeled hosts to form chimeric allantoises, no
triply labeled
(ß-galactosidase+/tritium+/benzidine+)
donor-derived erythroid cells were detected in the host allantoises following
24 hours of whole embryo culture (Downs et
al., 1998). Furthermore, those explants that were free-floating in
the exocoelom or fused only with the chorion and did not exhibit vascular
continuity with the fetus and/or yolk sac were entirely devoid of
benzidine-positive cells, suggesting that, in the mouse conceptus, the
allantois is not a source of erythroid cells
(Downs et al., 1998). However,
some data suggested that the murine allantois might have erythropoietic
potential: 5-10% of allantoises explanted between the neural plate and 4 s
stages (7.5-8.25 dpc), or cultured in isolation for 24 hours exhibited a
constant but relatively small population of erythroid cells, the origin of
which has never been accounted for (Downs
et al., 1998).
The aforementioned studies of the murine allantois were carried out during
only a short period (24 hours), and early markers of hematopoietic cells were
not examined. In light of recent experiments suggesting that hematopoietic
cells emerge from the placenta several days later
(Alvarez-Silva et al., 2003;
Gekas et al., 2005;
Ottersbach and Dzierzak,
2005), we undertook a series of experiments to examine the
hematopoietic potential of the allantois and the chorion prior to their fusion
and, more importantly, prior to the vascular link-up between the umbilical,
yolk sac and cardiovascular systems in the conceptus. First, we followed the
expression of Runx1 in both the intact conceptus and in explant cultures.
Runx1 is a transcription factor required for hematopoietic stem cell
formation, and is also an early marker for sites of hematopoietic cell
emergence in the embryo (Cai et al.,
2000; Ciau-Uitz et al.,
2000; Kalev-Zhylinska et al.,
2002; Lacaud et al.,
2002; North et al.,
2002; North et al.,
1999; Okuda et al.,
1996; Wang et al.,
1996). Runx1 expression has been documented in the
placenta (Ottersbach and Dzierzak,
2005), at the site of chorio-allantoic fusion
(North et al., 1999) and in
the chorion prior to fusion (Lacaud et
al., 2002), but has not been reported in the pre-fusion allantois.
Second, we cultured allantoises and chorions on OP9 stromal cells in the
presence of hematopoietic cytokines, or as explants on plastic followed by
methycellulose colony forming assays, to determine their hematopoietic
potential.
Here we show that both the allantois and chorion, having been isolated from early headfold-stage conceptuses before establishment of the circulation, express Runx1 and exhibit hematopoietic potential following explant culture. Furthermore, we show that this hematopoietic potential is not dependent on chorio-allantoic fusion, indicating that it is an intrinsic property of both tissues.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) with
Runx1lz/+ males (North
et al., 1999) (formal designations of the
Runx1rd and Runx1lz alleles are
Runx1tm1Spe and Runx1tm2Spe,
respectively). For marking experiments, B6.Cg-Tg(ACTB-Bgeo/GFP)21Lbe/J
(Jackson Laboratory, Bar Harbor, ME), Tg(Ly6a/GFP)
(Ottersbach and Dzierzak,
2005), or Runx1lz/+ male mice were bred to
female C57BL/6J, C57BL/6J x 129S1/SVImJ F1, or B6CBAF1/J mice (Jackson
Laboratory). We identified female mice in estrus, mated them 5 minutes before
the beginning of the dark cycle and then examined them 4 hours later for the
presence of a vaginal plug. Dissections, staging and morphological scoring
were performed as previously described
(Downs, 2006;
Downs and Davies, 1993;
Downs and Harmann, 1997;
Downs et al., 2004). All mouse
procedures were approved by our Institutions' Animal Care and Use
Committees.
Embryo dissection
We isolated allantoises by mouth aspiration
(Downs, 2006)
(Fig. 2A), and in some cases
subdivided the allantoises into distal, mid and proximal thirds with glass
scalpels.
To isolate the chorion, the Reichert's membrane was removed and the
embryonic and visceral endoderm was labeled by submerging the intact conceptus
for 1 minute in AlexaFluor594-conjugated Concanavalin A (ConA 594) [5 mg/ml in
phosphate-buffered saline (PBS)] (Molecular Probes/Invitrogen, Carlsbad, CA)
(Fig. 2B). The mesoderm lining
the exocoelomic cavity was labeled by injecting AlexaFluor488-conjugated
Concanavalin A (ConA 488) into the exocoelomic cavity using a mouth-held
microcapillary pipette. Tissues were digested using pancreatin/trypsin
(Downs, 2006) for 10 minutes
at 4°C and separated with the aid of glass scalpels and a mouth aspirator.
Separation of the chorion from visceral endoderm was confirmed by the absence
of ConA 594 and the presence of ConA 488 by fluorescence stereomicroscopy
(Leica, Ernst-Leitz-Strasse, Germany). Once this technique was established, we
labeled only the endoderm by dipping the conceptus into ConA 488 and removing
the ConA 488-positive tissue from the chorion.
We isolated PAS as described by Godin et al.
(Godin et al., 1993). We
isolated embryonic ectoderm by incubating headfold-stage conceptuses in ConA
488 to label the anterior head endoderm
(Fig. 2C). A transverse cut was
made below the amnion using a glass scalpel to separate the extraembryonic
from embryonic components of the conceptus. A sagittal cut was then made
through the epiblast; the anterior portion of the epiblast was saved. The
tissues were digested in trypsin/pancreatin at 4°C for 10 minutes and then
dissected using a glass needle and mouth aspirator to separate the anterior
neuro-ectoderm and mesoderm from the anterior head endoderm.
Explant cultures for Runx1 and Ly6A expression
Allantoises and chorions were cultured as explants directly on plastic (24
Well Cell Culture Cluster, Corning, Corning, NY) or in roller drum suspensions
for 48 hours (Downs, 2006).
Most material was at headfold stage, with a minor 1-2 s component. We fixed
the explants in 4% paraformaldehyde for 2 hours on ice; they were then washed
with PBS and stained for 6 hours for ß-galactosidase activity
(Miles et al., 1997). The
genotype of explants from Runx1lz/+ x
Runx1rd/+ matings was determined by PCR from corresponding
epiblast DNA using previously described primers and amplification conditions
(North et al., 1999).
OP9 explant cultures
Individual allantois, chorion, PAS, visceral endoderm and neuroectoderm
explants were added to a freshly prepared layer of OP9 stromal cells in single
wells of 12-well plates and grown in 1 ml -MEM (Gibco/Invitrogen)
containing 20% fetal bovine serum supplemented with stem cell factor (SCF, 50
ng/ml), interleukin 3 (IL3, 5 ng/ml), interleukin 6 (IL6, 5 ng/ml), Flt-3
ligand (Flt3-L, 10 ng/ml), granulocyte colony stimulating factor (G-CSF, 5
ng/ml), granulocyte monocyte stimulating factor (GM-CSF, 5 ng/ml) (all
cytokines from R&D Systems, Minneapolis, MN) and 2-mercaptoethanol (2-ME,
1x10-5 M) (Sigma) at 37°C in 5% CO2
(Nishikawa et al., 1998) to
encourage the proliferation and differentiation of myeloid lineage cells. Half
of the medium and cytokines were replaced every other day. Explant cultures
were analyzed for DiI-Ac-LDL (Biomedical Technologies, Stoughton, MA) uptake
or CD45 expression after 7 days of culture. Fluorescence activated cell
sorting (FACS) analyses were performed after 14 days of explant culture.
Flow cytometry
Single-cell suspensions were prepared by incubating the explants for 10
minutes in 500 µl of Cell Dissociation Buffer (Gibco/Invitrogen) at
37°C followed by passage through a 40-µm strainer. Explant cell
suspensions at a concentration of 2x106 cells per ml were
treated with Fc blocking serum (BD Pharmingen, San Diego, CA) for 20 minutes
at 4°C then incubated with CD45-PerCP (30-F11), Mac1-PE (M11/70),
c-kit-FITC (2B8), or Gr-1-FITC (RB6-85C) (BD Pharmingen or eBioscience, San
Diego, CA), washed and re-suspended in 200 µl FACS buffer (BioSure, Grass
Valley, CA). Dead cells were excluded with TO-PRO-3 (Molecular
Probes/Invitrogen), and cells were analyzed on a FACSCalibur flow cytometer
(Becton Dickinson, Rockville, MD) with FloJo software (Tree Star Inc.,
Ashland, OR).
Progenitor assays
Two-day explant cultures were performed by plating six conceptus
equivalents of allantoises or chorions in a single well of a 24-well plate
containing 500 µL of 50% rat serum/DMEM
(Downs et al., 2001). The
medium was replaced after 24 hours and at 48 hours the explants were washed in
PBS, then incubated in pre-heated (37°C) Dispase II (Roche, Basel,
Switzerland) for 25 minutes and mechanically disassociated using a pipette.
Cells were collected by centrifugation and cultured in 3.3 ml of 3434
Methocult (StemCell Technologies, Vancouver, Canada). Colonies were counted
and selected 7 days later.
Five-day explant cultures were performed by plating six conceptus equivalents of allantoises or chorions on OP9 cells in a single well of a 24-well plate in the presence of 50 ng/ml SCF, 10 ng/ml Flt3-L, 4 U/ml erythropoietin (EPO) and 1x10-5 M 2-ME to preferentially encourage the proliferation of erythroid progenitors. Explants were dissociated into single-cell suspensions as described above and plated in 3.3 ml of 3434 MethoCult (Stem Cell Technologies). Colonies were scored and harvested 10 days later.
ß-Globin expression analysis
RNA was extracted from individual burst-forming unit erythroid (BFU-E) or
colony-forming unit granulocyte-macrophage (CFU-GM) colonies, adult mouse bone
marrow, and 9.0 dpc mouse yolk sac using an RNA extraction kit (Qiagen,
Valencia, CA). 
-Globin (y) primers (5' TGT CCT CTG CCT CTG
CCA TAA 3' and 5' AGC GGA CAC ACA GGA TTG CTG 3')
(Fiering et al., 1995),
ß-major globin primers (5'-CTG ACA GAT GCT CTC TTG GG-3' and
5'-CAC AAA CCC CAG AAA CAG ACA-3') and Hprt primers
(5'GCT GGT GAA AAG GAC CTC T-3' and 5'-CAC AGG ACT AGA ACA
CCT GC-3') were used to analyze globin expression
(Keller et al., 1993). PCR was
performed for 29 cycles: 94°C, 30 seconds; 57°C, 30 seconds; 72°C,
45 seconds.
Histological analysis
Conceptuses were assayed for ß-galactosidase activity
(Miles et al., 1997), and 5
µm paraffin sections were either counterstained with Nuclear Fast Red
(Vector Labs, Burlingame, CA) or left unstained. Staining for
ß-galactosidase was performed for 6 hours at 37°C, unless otherwise
noted in the figure legends. We examined five conceptuses at bud stages, eight
at headfold stages, 17 at 1-16 s and two at 9.5 dpc.
Explants from OP9 cultures were incubated in 1 µl/ml DiI-Ac-LDL for 4 hours at 37°C, fixed in 4% paraformaldehyde, and then incubated first with CD45-biotinylated antibody (BD Pharmingen) overnight at 4°C and then with Pacific Blue-conjugated streptavidin (Molecular Probes/Invitrogen) for 90 minutes at room temperature. Explants were observed by fluorescence stereomicroscopy and photographed using a monochrome digital camera (Roper Scientific, Tucson, AZ).
Single-cell suspensions of explants (1x104 cells) or individually selected colonies were centrifuged onto slides (14 g, 8 minutes), dried for 3 hours at room temperature and stained by May-Grunwald Giemsa. Cytospin preparations were observed by compound microscopy and photographed using a digital color camera (Diagnostic Instruments, Sterling Heights, MI).
All explant experiments were performed at least three times on headfold-stage material.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7.5-9.25 dpc)
(Fig. 1). We and others
previously reported that at stages prior to fusion, Runx1 is expressed in yolk
sac visceral endoderm (North et al.,
1999), the vascular and hematopoietic components of yolk sac blood
islands (Lacaud et al., 2002;
North et al., 1999), and in
chorionic mesoderm (Lacaud et al.,
2002). We observed Runx1 expression, as visualized from a lacZ
`knock-in' allele (North et al.,
1999), in these same locations
(Fig. 1B). Runx1 expression in
the chorion was first seen as punctate blue staining in some mesodermal cells
beginning at the early bud stage (not shown), and was intense in most
chorionic mesodermal cells by the late headfold stage
(Fig. 1B). Chorionic ectoderm
was negative at all stages examined. In the allantois, we found convincing
blue stain within the proximal allantois and amnion where these two tissues
join, as well as faint blue stain within the distal portion of the allantois,
including the outer layer of mesothelium in some specimens (not shown). After
chorio-allantoic fusion (12 s, 9.0 dpc), Runx1 expression was seen within
internal cells at the chorio-allantoic interface
(Fig. 1C-E), at external sites
where the allantois intersects the chorion
(Fig. 1E, blue arrows) and also
in a small cluster of cells within the base of the allantois
(Fig. 1E,F). At later times
(16 s, 9.25 dpc), Runx1 expression was seen both at the plane of
chorio-allantoic fusion (Fig.
1G) and in endothelial cells lining the proximal allantoic
vasculature (Fig. 1G,H) but not
in the mid-region of the allantois (Fig.
1G). Runx1 expression in allantoic mesothelium was clearly seen in
the 9.5 dpc conceptus (Fig.
1I,J). By 10.5 dpc, the umbilical artery, which forms from
the allantois, expressed Runx1 throughout its length both in endothelial cells
and in intra-aortic hematopoietic clusters
(Fig. 1K,L) as previously
described (North et al.,
1999). In previous studies with the same
Runx1lz allele, Ottersbach and Dzierzak
(Ottersbach and Dzierzak,
2005) showed that by 11.0 dpc, Runx1 is expressed in the
labyrinth region of the placenta in endothelial cells, in cells underlying the
endothelium and in cells within the circulation.
Faint, punctate blue staining was observed in both endodermal and
mesodermal components of the visceral yolk sac as previously described
(North et al., 1999) (not
shown), and in extra-embryonic visceral endoderm overlying the chorionic
ectoderm [`distal' extraembryonic endoderm or `ectoplacental' endoderm
(Duval, 1891)]
(Fig. 1B).
Runx1 is expressed in both the allantois and the chorion, independent of their union
Although our results of histology clearly revealed Runx1 expression in the
chorion prior to chorio-allantoic fusion, allantoic mesothelial expression was
much less intense. In addition, once chorio-allantoic fusion has occurred it
is no longer possible to determine whether the Runx1+ cells at the
fusion junction originated from the allantois, from the chorion, or from both
tissues. Therefore, to confirm that both the allantois and chorion express
Runx1, we isolated both tissues prior to their fusion, cultured them in vitro
as explants and analyzed the explants for Runx1 expression.
|
). When aspirated via a mouth-held microcapillary pipette, the
entire allantois can be removed from these insertion sites. We aspirated
allantoises from early headfold to 1-2 s conceptuses with a glass pipette
(Fig. 2A). Following
aspiration, we carefully trimmed several cell layers from the base of the
allantois with a glass scalpel to ensure that the allantoises were not
contaminated with yolk-sac-derived cells. Allantoises from
Runx1lz/+ conceptuses were then cultured for 48 hours on
plastic, after which time the explants were assayed for ß-galactosidase
activity. We estimated that 48-hour explants were the equivalent of 9.75
dpc (20 s), as allantoises appear to have an internal timing mechanism
revealed by their normal behavior even when removed from contact with the
primitive streak (Downs and Gardner,
1995; Downs et al.,
2004; Downs et al.,
2001). Plated allantoic explants gave rise to Runx1+
cells (Fig. 3A).
Runx1+ cells included cells that remained within the explant, as
well as mesenchymal-like cells that had crawled off the explant
(Fig. 3A). These motile cells
were previously shown by limited fate mapping to originate within the
allantoic mesothelium (Downs et al.,
2004), confirming that at least some of the Runx1+
cells came from the mesothelial component of the allantois.
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9.75 dpc). Runx1 expression was
observed in the spheres that formed under these culture conditions
(Fig. 3C). Sectioning through
the spheres revealed that Runx1 was expressed in the outer one to two cell
layers around the surface of the spheres
(Fig. 3C), consistent with
expression in mesothelium. To discover which region of the allantois gave rise
to Runx1+ cells, we also subdivided headfold-stage allantoises into
distal, mid and proximal thirds and cultured these pieces separately as 24- or
48-hour explants on tissue culture plastic. All three pieces of the allantois
gave rise to Runx1+ cells (not shown), indicating that Runx1
expression may ultimately initiate throughout the length of the allantois. To assess whether the chorion also expressed Runx1, we isolated chorions and cultured them as explants for 48 hours on tissue culture plastic (Fig. 3B). Runx1+ cells persisted in chorionic explants, and most had crawled off the explant. As we did not separate chorionic mesoderm from ectoderm, we do not know which chorionic component gave rise to the Runx1+ cells in the explant culture, but given that chorionic mesoderm is Runx1+, and that mesoderm is characteristically migratory, we suspect that the plated Runx1+ population was derived from chorionic mesoderm.
In addition to Runx1, we also analyzed the expression of another
hematopoietic marker, Sca1, as visualized with a Ly6a/GFP transgene
(de Bruijn et al., 2002;
Ottersbach and Dzierzak, 2005)
in allantoises and chorions before and after explant culture. Ottersbach and
Dzierzak (Ottersbach and Dzierzak,
2005) reported that Ly6a/GFP is expressed in the extra-embryonic
ectoderm and ectoplacental cone beginning in pre-streak-stage conceptuses.
Consistent with their findings, we observed Ly6a/GFP expression in the freshly
isolated chorion but not in the allantois
(Fig. 3D). After 24 hours of
explant culture, both the allantois and the chorion expressed Ly6a/GFP
(Fig. 3D).
Thus, on the basis of these observations, we conclude that Runx1, one of the earliest markers of definitive hematopoiesis, is expressed in both the allantois and in the chorionic mesoderm. Furthermore, Runx1 expression in the allantois and the chorion is independent of fusion and is therefore intrinsic to these two tissues.
The allantois and chorion have hematopoietic potential
We next assessed the hematopoietic potential of allantoises and chorions
isolated from early headfold to 1-2 s conceptuses, before a circulatory
continuum is established between the major vascular systems in the conceptus.
We first cultured allantoic and chorionic explants on OP9 stromal cells
(Suzuki and Nakano, 2001) in
the presence of hematopoietic cytokines that support the growth and
differentiation of myeloid lineage cells (IL3, IL6, G-CSF, GM-CSF). Both the
allantois and chorion from wild-type conceptuses gave rise to abundant round
nonadherent cells (Fig. 4C,E)
at efficiencies that were equivalent to those seen with the PAS, an
established hematopoietic territory (Table
1). In contrast, allantoises and chorions isolated from
Runx1-deficient conceptuses failed to yield round nonadherent cells
(Fig. 4D,F), consistent with
the strict requirement for Runx1 in establishing definitive hematopoiesis
(Okuda et al., 1996;
Wang et al., 1996).
|
;
Li et al., 2006;
Mukouyama et al., 2000;
Okuda et al., 1996;
Wang et al., 1996). Cells
expressing CD45, c-kit, or both antigens were detected in nonadherent cells
collected from both the allantois and chorion explants from wild-type
conceptuses (Fig. 4K), while
cells expressing these markers were absent in cultures from Runx1-deficient
conceptuses. Many of the CD45+ cells generated from allantois and
chorion explants cultured in SCF, IL3, IL6, Flt3-L, G-CSF and GM-CSF expressed
either or both the myeloid cell markers Gr-1 and Mac-1
(Fig. 5A), and cytospin
preparations accordingly revealed myeloid lineage cells in the cultures
(Fig. 5B).
As a second approach we performed hematopoietic progenitor assays following
5 days of OP9 explant culture in the presence of SCF, Flt-3 ligand and EPO,
while omitting G-CSF, GM-CSF, IL6 and IL3 in order to preferentially encourage
the proliferation of erythroid progenitors. We found that granulocyte,
erythrocyte, monocyte, megakaryocyte (CFU-GEMM), granulocyte-macrophage
(CFU-GM) and erythroid (BFU-E) progenitors were present in the explant
cultures (Fig. 5C).
Hematopoietic progenitors were also found in explants following 2 days of
culture on plastic in 50% rat serum (Fig.
5C); they appeared at the same time and under the same conditions
as the expression of Runx1 in the explants
(Fig. 3A,B). We analyzed
ß-globin gene expression in individual colonies by RTPCR to determine
whether the BFU-E colonies contained primitive or definitive erythroid cells.
The erythroid colonies expressed ß-major globin but not y, the
latter of which is a specific marker of primitive, yolk-sac-derived erythroid
cells (Brotherton et al., 1979;
Wong et al., 1983), indicating
that the BFU-E colonies consisted of definitive erythroid cells.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
By 11.0 dpc, the placenta is also a source of long-term repopulating
hematopoietic stem cells (Gekas et al.,
2005; Ottersbach and Dzierzak,
2005). However, whether these cells were derived from other
hematopoietic sites within the conceptus, such as the yolk sac and
aorta-gonad-mesonephros (AGM) region, or from the allantois and/or chorion
themselves was not known. In this study, we have demonstrated that both the allantois and the chorion have hematopoietic potential. The potential of both tissues was revealed after culturing them as explants on OP9 stromal cells in the presence of hematopoietic cytokines, or by CFU-C assays following a 2 day explant culture on plastic. Explants formed myeloid lineage cells and progenitors for definitive erythroid cells whereas Runx1-deficient explants did not. Thus, our results strongly suggest that Runx1 expression identifies hematopoietic progenitor cells within both the allantois and chorion of the intact conceptus.
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;
Downs and Harmann, 1997).
However, ectopically transplanted allantoic cells from the distal, middle and
proximal portions of the allantois did contribute extensively to the
endothelium of the dorsal aorta and the mesenchyme directly adjacent to it
within the PAS of the host embryo (Downs
and Harmann, 1997). The PAS is an established intra-embryonic
hematopoietic territory and, between the headfold and 10 s stages, contains
multipotent hematopoietic activity that could be revealed following explant
culture on stromal cells (Cumano et al.,
1996; Cumano et al.,
2001). In retrospect, the extensive contribution of the allantois
to the dorsal aorta and periaortic mesenchyme upon transplantation
(Downs and Harmann, 1997) is
consistent with the hematopoietic potential demonstrated in vitro in the
current study. Importantly, the hematopoietic potential of the allantois was
demonstrated here in all three allantoic subregions of early headfold stage
conceptuses, before vascular continuity between the allantois and yolk sac was
achieved. Therefore the hematopoietic progenitors must have arisen de novo
from the allantois and not via the circulation from the yolk sac or the embryo
proper. Although we did not separate the chorionic mesoderm from ectoderm, we suspect, based on our results of ß-galactosidase staining, that the Runx1+ mesodermal component is the source of the hematopoietic cells supplied to the chorion. The site within the allantois that gives rise to hematopoietic cells is not clear; however, as Runx1+ cells were located on the outer mesothelial surface, which mediates chorio-allantoic union, it is likely that the allantoic mesothelium is involved, with Runx1+ endothelial cells within the base of the allantois being a second potential source.
Very little is known about either allantoic mesothelium or chorionic
mesoderm other than that they mediate chorio-allantoic fusion
(Downs and Gardner, 1995) via
VCAM1 on the allantoic mesothelium
(Gurtner et al., 1995;
Kwee et al., 1995) and its
counter receptor 4 integrin on chorionic mesoderm
(Yang et al., 1995). Once the
allantois fuses with the chorion, it is not known whether the fusing surfaces
of the allantois and chorion break down, persist or differentiate into other
cell types (Downs, 2002).
Based on findings presented here, we suggest that cells from one or both of
these fusing surfaces persist. In this way, Runx1+ hematopoietic
cells derived from allantoic mesothelium and/or chorionic mesoderm may then
enter the allantoic vasculature during or after its penetration into the
chorionic plate. One interesting observation is that Runx1 expression in the
chorionic mesoderm and at the site of intersection between the allantois and
the chorion very closely resembles that of 4 integrin, both temporally
and spatially (Downs, 2002).
Both 4 integrin and Runx1 are expressed on all long-term repopulating
hematopoietic stem cells in the embryo
(Gribi et al., 2006;
North et al., 2002), lending
support to the notion that the Runx1+ and 4
integrin+ cells at the chorio-allantoic fusion junction ultimately
contribute to hematopoiesis in the placenta.
One of the issues raised by these studies concerns the location of the
source of the signal (or signals) that induces the hematopoietic program in
the allantois and chorion. Studies in both avian and murine conceptuses
clearly show that the induction of the hematopoietic program in mesoderm
requires a signal from endoderm. For example, in the mammalian yolk sac,
visceral endoderm provides soluble signals that are required for the formation
of blood islands (Belaoussoff et al.,
1998). In the avian embryo, a brief exposure to endoderm can
re-specify axial and somatopleural mesoderm, which has no hematopoietic
potential, to become splanchnopleural mesoderm that could contribute to the
formation of hematopoietic cells in the dorsal aorta
(Pardanaud and Dieterlen-Lièvre,
1999). The endodermal signal in the avian allantois was thought to
originate from the core of endoderm within the allantois proper. The allantois
of avian embryos emanates from the intra-embryonic splanchnopleural mesoderm
and contains within it endoderm derived from the primitive hindgut
(Caprioli et al., 1998).
However, whereas the allantois of many eutherian mammals exhibits an
endodermal component (Mossman,
1937), the allantois of the house mouse is thought to consist
entirely of extraembryonic mesoderm that contains within it a core of
Brachyury-positive cells and is surrounded by a mesoderm-derived mesothelial
layer (Duval, 1891;
Inman and Downs, 2006;
Mossman, 1937). A
re-examination of the murine allantois for an endodermal component using
modern techniques and markers may be warranted.
The distal tip of the allantois is the `oldest' allantoic tissue, having
emerged first from the primitive streak
(Downs et al., 2004;
Kinder et al., 1999;
Lawson et al., 1991), whereas
the cluster of cells within the base of the allantois emerges later
(Kinder et al., 1999). We
hypothesize that signals separated either temporally or spatially may activate
Runx1 expression in the distal portion of the allantois and within the base.
It is tempting to speculate that, as the mesoderm at these two sites
originates within the primitive streak over a prolonged period (6.75-8.5 dpc)
(Beddington, 1982;
Copp et al., 1986;
Downs, 2002;
Kinder et al., 1999;
Lawson et al., 1991;
Tam and Beddington, 1987),
these signals may be encountered at different times in the primitive streak.
However, as so little is known about the role of the streak in specification
of allantoic mesoderm (Downs et al.,
2004), further experimentation is needed to address this notion.
Moreover, as little is known about chorionic mesoderm and ectoderm, the
signals responsible for Runx1 expression in the chorionic mesoderm are
completely unclear at this time.
In summary, our findings strongly suggest that the placenta itself is a hematopoietic organ. To what extent hematopoietic activity emerges de novo from the placenta, or comes from other sites of hematopoietic emergence awaits further investigation.
|
ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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