Extraembryonic Origin of Circulating Endothelial Cells
Luc Pardanaud1,2,3*, Anne Eichmann1,2,3¤
1Center for Interdisciplinary Research in Biology (CIRB), Colle `ge de France, Paris, France, 2INSERM U1050, 75005 Paris, France, 3CNRS UMR 7241, 75005 Paris, France
Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate
to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at
improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been
addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature.
CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac
chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk
sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes.
Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the
embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories
made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are
of intra-embryonic origin.
Citation: Pardanaud L, Eichmann A (2011) Extraembryonic Origin of Circulating Endothelial Cells. PLoS ONE 6(10): e25889. doi:10.1371/journal.pone.0025889
Editor: Costanza Emanueli, University of Bristol, United Kingdom
Received June 30, 2011; Accepted September 12, 2011; Published October 14, 2011
Copyright: ? 2011 Pardanaud, Eichmann. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Inserm, Fondation pour la recherche me ´dicale (FRM) and Fondation Bettencourt-Schueller. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤ Current address: Department of Cardiology, Yale University School of Medicine, New Haven, Connecticut, United States of America
The existence of circulating endothelial cells (CEC) was
demonstrated ten years ago in adult mice [1,2]. They were shown
to reside in the bone marrow and were mobilized during
physiological and pathological angiogenesis including tumor
growth and heart failure [3,4]. These findings opened large
perspectives and hopes concerning the use of these cells as putative
tools to specifically target tumors or damaged tissues. Clinical trials
using marrow-derived CEC injected into patients with coronary
heart disease have led to a mild improvement in left ventricular
ejection fraction and myocardial perfusion [5–8], indicating that
CEC hold promise for the treatment of disease, but that their
contribution to healing of damaged tissue remains to be studied
The role of CEC during angiogenesis is debated in the literature
: while some studies have shown that CEC are actively
mobilized during tumor angiogenesis, integrate the lumen of
neovessels  and play a role in metastasis dissemination ,
other authors showed that CEC mobilization during tumorigenesis
is inefficient  and that these cells rarely reach tumor vessels but
rather form a niche of bone-marrow-derived hematopoietic
progenitors that colonize the vascular wall and contribute to
angiogenesis by release of soluble factors [13–16]. A recent review
on targeted cancer gene therapy using CEC concluded that
although feasible, the efficacy of this strategy to control tumor
burden has not shown overwhelming success .
The true identity of CEC in vivo also still remains uncertain ten
years after their discovery. In particular, diverse molecular
markers have been used to describe various forms of CEC and
progenitors, here collectively referred to as CEC, including bone
marrow-derived endothelial progenitor cells (EPC), cord blood-
derived EPC, high proliferative potential-endothelial colony
forming cells (ECFC), low proliferative potential-ECFC, endothe-
lial outgrowth cells or mesenchymal stem cells . The diversity
of adult CEC suggested that they might have multiple potential
sites of origin, similar to hematopoietic stem cells (HSC).
Alternatively, they might originate from a single source and
acquire diversity at later stages.
All known types of CEC share expression of at least some
molecular markers with endothelial cells (EC) and hematopoietic
cells (HC), reflecting the close developmental relationship between
these cell types. In the adult bone marrow and in the embryo, EC,
CEC and HSC reside together in stem cell niches. During
embryonic development, EC and HC are first formed as
‘hemangioblastic clusters’ of tightly associated precursors in the
yolk sac blood islands [19,20]. Yolk sac hemangioblasts generate a
transient wave of extra-embryonic EC and circulating HC of the
erythropoietic and macrophage lineages. This transient first wave
of HC is later replaced by an intra-embryonic source of definitive
HSC , defined by their ability to repopulate lethally irradiated
hosts after transplantation . Definitive HSC are morpholog-
ically conspicuous in the ventral wall of the dorsal aorta, where
they bud off the endothelial lining and give rise to HSC . The
epithelio-mesenchymal transformation of these ‘hemogenic’ EC is
enhanced by shear stress generated by the blood flowing through
the aorta and requires NO-production [23–25]. Thus, while the
yolk sac generates a transient population of EC and HC, definitive
HSC are born in close contact to the endothelium of the dorsal
aorta, in an intra-embryonic location.
The embryonic site(s) of CEC production had not been
determined previously. Using a parabiosis model between chick
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and quail embryos, we had previously shown that CEC are
produced in the embryo prior to bone marrow formation . We
had moreover shown that embryonic CEC show hallmarks of
adult CEC identified in mouse models, in that they are rare cells
sometimes integrated in vessels but mostly located in the
interstitium, which participate to neo-angiogenesis processes
including wound healing.
We here asked if CEC are first produced in the yolk sac and/or
have a double yolk sac as well as intra-embryonic origin. To
distinguish between these possibilities, we used a quail-chick
chimera model where chick embryos are grafted onto a quail yolk
sac and vice-versa. This model was instrumental to demonstrate
the intra-embryonic origin of definitive HSC 30 years ago .
Quail EC, as well as CEC, can be specifically labeled with the
monoclonal antibody QH1 . We found that the yolk sac, but
not the embryo proper, gives rise to CEC. Furthermore,
constructing half embryo chimeras in which the caudal part of
the embryo was of quail origin and the rest of chick origin, we
found that the allantois, previously shown to generate CEC when
ectopically grafted , is also able to give rise to CEC in situ.
Thus, using these models, we show here that CEC are generated
exclusively in extraembryonic territories made with splanchno-
pleural mesoderm and endoderm, but not in the embryo proper.
Materials and Methods
Yolk sac chimeras
Chick (Gallus gallus, JA57) and quail (Coturnix coturnix
japonica) eggs were incubated horizontally for 24h at 38uC until
they developed the appropriate somitic stages (s.s.). Yolk sac
chimeras  were made with a donor and a host at the same s.s.
and only embryos that had not yet established circulation (younger
than 13s.s.) were used. The donor embryo was isolated together
with its yolk sac and cleaned in phosphate buffered saline
(PBS)+Ca2+Mg2+. Using Pascheff scissors the embryo was carefully
separated from the yolk sac at the level of the margin separating the
two regions. The host was injected with Indian ink (diluted 1/1 in
PBS+Ca2+Mg2+) beneath the blastoderm, and the embryonic
territory was removed from the egg. Then the donor embryo was
positioned at the surface of the host egg. A crucial point to ensure
survival of grafts was to prepare a donor blastodisc just larger than
hole left after removal of the host embryo to avoid leakage of yolk.
The donor embryo was placed at the top of the hole in the proper
orientation and the hostandthedonortissues werecarefullysutured
at their margin using two pairs of fine forceps. The string of excess
tissues at the surface of the suture was removed with Pascheff
scissors, avoiding any leakage around the suture (Fig. 1A). The egg
was then sealed with scotch tape and re-incubated for 1 to 13 days
(Fig. 1B–F). Both possible combinations, quail embryo on chick yolk
sac and chick embryo on quail yolk sac, were generated. Until E6.5,
embryos with a part of the yolk sac were fixed overnight at 4uC in
Paraformaldehyde 4% (PAF) or Alcohol 100u/Acetic Acid 1% at
220uC, dehydrated, embedded in paraffin, serially sectioned and
stained. In older chimeras, pieces of choriallantoic membrane
(CAM), meninges, mesentery, aorta, jugular veins and skin were
isolated and fixed in PAF. All were processed for in toto
immunostaining after overnight PAF fixation as previously
described . Bone marrow was retrieved by dissociating long
bones. Drops (20ml) of bone marrow cellular suspension in culture
medium (DMEM+10% Fetal Calf Serum-FCS-) were placed in
Petri Dishes (35mm) and cultured 24h at 37uC, 5% CO2. Cultures
were fixed in PAF 15mn at room temperature, rinsed in PBS and
processed for immunocytochemistry.
Wound healing assays
On E5 yolk sac chimeras, the chick amnion was sectioned and a
wing was exposed at the surface. A deep longitudinal incision was
made at the tip of the wing using a microscalpel. The embryos
were sacrificed after 3 to 8 hours (n=20). The two chick wings
(wounded and contralateral) were isolated, fixed in Alcohol 100u/
Acetic Acid 1% at 220uC, dehydrated, embedded in paraffin,
serially sectioned and stained.
In vitro quail-chick chimeras
As above, pre-circulation quail and chick embryos at the same
stage (5–12s.s.) were used. A chick blastodisc, isolated together
with its yolk sac was placed in a Petri dish, on a semisolid medium
containing 50% agar, 25% yolk, 20% PBS+Ca2+Mg2+and 5%
penicillin-streptomycin. A quail embryo was carefully isolated
from its yolk sac and placed in the Petri dish, close to the chick
yolk sac. Using two pairs of fine forceps, a suture was made
between the quail and chick territories. The embryos were
incubated during 1 to 2 days at 37uC, 5%CO2 then fixed in
Alcohol 100u/Acetic Acid 1% at 220uC, dehydrated and
embedded in paraffin.
Half embryo chimeras
The technical approach was the same as above except that we
isolated the territory behind the last formed somite from the quail
embryo and grafted it into a chick recipient from which the
corresponding piece had been removed. The chimeras were
incubated until E5.5–E6, a stage where the allantois was well
differentiated. Chimeras, allantois and a part of yolk sac were
separately fixed in PAF, dehydrated, embedded in paraffin, serially
sectioned and stained.
Different double/triple stainings were performed using the
QH1  immunostaining was performed on 7.5 mm paraffin
sections or cells as previously described . QH1 (undiluted
hybridoma supernatant or 1/1000e ascites -Developmental
Studies Hybridoma Bank -DSHB- developed under the auspices
of the NICHD and maintained by The University of Iowa,
Department of Biology, Iowa City, IA 52242) was visualized using
peroxydase- (BioRad) or Alexa 350/488/555- (Invitrogen) conju-
gated secondary antibodies.
BEN monoclonal antibody (DSHB) stains quail and chick
peripheral projecting neurons and hematopoietic precursors .
After rehydration, sections were pretreated with 0.025% trypsin at
37uC for 30 minutes. BEN (1/10e) was revealed by an Alexa 488/
555 goat anti mouse IgG1 (Invitrogen).
LEP100 monoclonal antibody (DSHB) stains avian macrophag-
es . After an incubation in PBS/FCS 3%/Triton 0.1%,
LEP100 (1/5) was revealed by an Alexa 488/555 goat anti mouse
8F3 monoclonal antibody (DSHB) stains the cytoplasm of all
chick cells but does not stain quail cells . 8F3 (1/5) was
revealed by an Alexa 488 goat anti mouse IgG1 (Invitrogen).
Polyclonal Rabbit Anti-Human von Willebrand Factor (DakoCy-
sections were pretreated with 0.025% trypsin at 37uC for 30 minutes.
The antibody was diluted 1/100 in PBS/triton 0.1% and was
revealed by an Alexa 555 goat anti rabbit (Invitrogen).
Biotinylated Sambucus nigra lectin (1/400 in PBLEC buffer-PBS
pH6.8, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM MnCl2, 1% triton)
recognizes chick and quail EC (Clinisciences, ) but also avian
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macrophages. The lectin was revealed using Cy3 streptavidin
Biotinylated LEA agglutinin (Lycopersicon esculentum, Sigma) labels
avian macrophages and venous endothelium . After rehydra-
tion, sections were pretreated with 0.025% trypsin at 37uC for
10 minutes. Biotinylated LEA (20 mg/ml in PBS-0.1% triton,
overnight at 4uC) was revealed using Cy3 streptavidin (Amer-
Nuclei were counterstained with glychemalun or Hoechst 33342
after the immunochemistry .
Quantification of QH1+CEC
Observation and counting was done using Leica or Olympus
microscopes. For each harvested graft, serial transverse sections
were prepared. The volume of embryos or wing buds was
calculated using a micrometric scale by measuring the surface of
the first section where the tissue was visible plus one section per
slide multiplied by the thickness of all sections containing
embryonic tissues (n67.5 mm). The number of quail QH1+EC
was counted manually on 1150 sections (x25 objective, final
magnification x110, 2300 counted cells). For cell counting in bone
marrow cultures, the percentage of 8F3+cells was calculated on 10
different randomly chosen fields per chimera. Statistical analyses
were carried out using Mann-Whitney’s test.
Yolk sac origin of CEC
To determine if the yolk sac produces CEC we created yolk sac
chimeras in which pre-circulation chick embryos are grafted on
Figure 1. Generation of yolk sac chimeras. A–C) chick embryos on quail yolk sacs; A) A yolk sac chimera just after the operation: in this case, a 10
somite old chick embryo (C) is grafted on a quail yolk sac recipient at the same stage (Q). The black arrowheads show the sinus marginalis, the limit of
the quail vascular area. The white arrowheads point to the suture between the chick and quail territories. The asterisk marks the chick head. Bar:
1 mm. B) A yolk sac chimera two days after the operation: in the quail egg a chick embryo (CE) correctly develops and vascular connections with the
quail yolk sac (QYS) are normal. Bar: 2 mm. C) A chimera after four days, with a healthy E5.5 chick embryo (CE), wrapped in the amnion (arrowhead)
and connected to a quail yolk sac (QYS). Bar: 1.5 mm. D–F) quail embryos on chick yolk sacs; D) After 5 days the quail embryo (QE) developed in
connection with the chick yolk sac (CYS). The arrow points to a CAM vessel. Bar: 2 mm. E) An E13 quail embryo (QE) wrapped in its CAM (arrow)
develops on a chick yolk sac (CYS). Bar: 7 mm. F) An E15 quail chimera isolated from the chick egg. Bar: 8 mm.
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yolk sacs from quail embryos at the same s.s. (Fig. 1A, see
Methods). As this graft involves replacement of an entire embryo,
without damaging the yolk sac of the host, it is technically very
challenging, but uniquely suited to study the developmental
potential of the yolk sac to generate CEC . Following the onset
of blood-flow, cells can circulate from the yolk sac to the embryo,
and CEC originating from the quail yolk sac can be traced using
the QH1 monoclonal antibody , which specifically recognizes
quail EC and HC. QH1 also labels non-circulating quail EC in the
yolk sac of the chimeras and HC, in particular yolk sac derived
macrophages. Quail CEC were distinguished from macrophages
using LEA and LEP100 macrophage-specific markers [31,34]:
quail macrophages were QH1+/LEA+or QH1+/LEP100+while
quail CEC were only recognized by QH1 (Fig. 2 A–C).
Furthermore quail CEC were identified using specific endothelial
markers vWF and Sambucus nigra [26,34] in addition to QH1.
One hundred and one yolk sac chimeras were constructed
(Fig. 1A). Their survival was 38% after one day, 38% after two
days and 32% after four days. The oldest embryos we obtained
reached 5.5 days of development (stage 28 of Hamburger and
Hamilton, HH, ). Beyond this stage, all chick embryos (n=9)
died within 12h, probably because the quail yolk sac did not grow
large enough to feed the chick embryos, which dramatically
increase in size from E5 onwards.
Serial sections of 27 chimeras were prepared, 3 after one day, 2
after two days (Fig. 1B) and 22 after four days (Fig. 1C). In all
cases, the chick embryos developed normally in the quail eggs, the
morphology and the blood supply was comparable to un-operated
embryos and the suture between the chick tissues and the quail
yolk sac had become invisible (Fig. 1B, C). The histology of chick
embryos also confirmed normal morphogenesis and organogen-
esis. Numerous yolk sac derived QH1+/LEA+macrophages
rapidly invaded chick tissues in particular in the head, the limb
buds and around the aorta (Fig. 2A). Their morphology was
variable, most cells being round (Fig. 2B, C) but in some cases
macrophages appeared as thin long cells or star shaped cells (not
shown). Most QH1+/LEA+macrophages remained isolated in
tissues (Fig. 2A–C) but formed aggregates in some cases (not
shown). Chick QH12/LEA+macrophages were also identified
Yolk sac-derived CEC colonized the chick tissues as soon as the
first day after surgery (Fig. 2). Their number was much less
important than the number of invading macrophages (Fig. 2D–H).
Quantification showed that the total number of quail CEC
increased from day 1 to day 4 after operation (from 192 to 517
cells/embryo, Fig. 2I) but that their density per mm3of embryo
decreased with time (from 143 to 26 cells/mm3at day 4 after
operation). The density of CEC found at day 4 after operation is
equivalent to that previously observed in quail-chick parabioses
before the onset of bone marrow formation , suggesting that
most CEC are generated in the yolk sac (Fig. 2I).
Quail yolk sac CEC were distributed throughout the chick
embryos at all stages analyzed; they colonized the meninges and
the periphery of the neural tube (Fig. 2B, E), the somatopleural
and splanchnopleural mesoderm, the dermis (Fig. 2F), myotome
and the dorsal root ganglia (Fig. 2G). During organogenesis, quail
CEC were found in the heart (Fig. 2C), the liver, the lung and the
limb buds (Fig. 2H). The proportion of interstitial (Fig. 2B, D) and
endothelium-integrated (Fig. 2C, E–H) quail CEC was quite
similar. Within the endothelium, quail CEC were mostly localized
in capillaries and exceptionally in large vessels such as the aorta or
the cardinal vein (not shown). The contribution of CEC to the host
endothelium was assessed by counting the percentage of QH1+
CEC inserted in chick endothelium on 9 E5 yolk sac chimeras
(chick embryo on quail yolk sac). The percentage of CEC that
participated to host endothelium was 61% (Fig. 2J), as shown
previously for parabioses .
To study later stages, in particular the bone marrow, we
performed grafting experiments in the reverse combination, quail
embryo on chick yolk sac. In all cases, the quail embryos
developed normally in the chick eggs, with a morphology and a
blood supply similar to un-operated embryos and the suture
between the chick tissues and the quail yolk sac was invisible
(Fig. 1D). In this combination, the survival of quail embryos was
possible beyond five days after the operation, most likely because
the larger size of the chick yolk sac allowed sufficient blood and
nutrient supply to sustain growth of the quail embryo (Fig. 1E, F).
The resulting chimeras (5/33) were autopsied when they
reached E13 (1/5, Fig. 1E)–E15 (3/5, Fig. 1F). One chimera
was left up to E15 to hatch. The embryo was perfectly developed
but did not manage to hatch, probably because the chick shell was
too thick to break for the quail embryo (not shown).
On 4 of these chimeras, quail bone marrow was collected from
long bones and cultured overnight before fixation. In culture,
fibroblasts, red blood cells and adipocytes were easily identified
with bright field observations (Fig. 3A). If CEC were produced in
the yolk sac, these chimeras should contain chick CEC in the quail
bone marrow. To identify chick cells, we used the 8F3 monoclonal
antibody specific for the cytoplasm of all chick cells (Fig. 3B, D, F,
S1A, B). The percentage of chick cells in the bone marrow,
calculated using 8F3/Hoechst double staining, was around 2%
(Fig. 3E). Bone marrow cultures and bone marrow smears indeed
contained 8F3+/vWF+(Fig. 3B) and QH12/vWF+(Fig. 3C, D)
cells, attesting the presence of chick CEC in the quail bone
marrow. As expected, other chick cells were present as well.
To characterize 8F3+cells, different double stainings were
performed. 8F3/LEA double staining showed that some chick cells
were not macrophages (Fig. S1B). QH1/BEN double staining
permitted to show that the major part of QH1+cells were
hematopoietic progenitors (Fig. S1C). QH1+/vWF+as well as
QH1+/BEN2cells (Fig. 3C, S1C) were probably quail EC coming
from sinusoids present in the bone marrow. Nevertheless, we
cannot exclude they were quail CEC coming from an unknown
Finally, the analysis of blood smears taken from host quails
permitted to find QH1+HSC (Fig. 3F) and to identify a few
QH12/8F3+/vWF+chick CEC (Fig. 3G).
We also histologically checked the presence of chick CEC in
organs. As the 8F3 antibody did not work on paraffin section in
our conditions, we analyzed sections with QH1/LEP100/
Sambucus nigra staining which permitted to recognize QH12/
LEP 1002/Sambucus+chick CEC. The CEC were found either
integrated in vessels (Fig 3H) or interstitially located (Fig. 3I, J).
The various observations made on later developmental stages
confirmed the ability of the yolk sac to produce CEC.
The organs were also invaded by chick macrophages. In the
meninges, in toto QH1/8F3 immunostaining showed a lot of 8F3+
chick cells located close to QH1+capillaries of the pia mater (Fig.
S2A). At higher magnifications, 8F3+filopodia projecting towards
QH1+capillaries were sometimes observed (Fig. S2B). On section,
a QH1/Sambucus/LEP100 staining confirmed the macrophage
identity of these cells (QH12/Sambucus+/LEP100+, Fig. S2C). In
the skin, chick cells were identified as macrophages (Fig. S2D, E).
Chick macrophages were also observed in internal organs as the
heart (Fig. S2F). In toto immunostainings showed that no 8F3+
chick cells were present in large vessels (aorta and jugular vein) as
well as in aortic vasa vasorum (Fig. S2G–I).
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Figure 2. Identification of yolk sac-born QH1+cells in E5.5 chick embryos isolated from yolk sac chimeras. Transverse sections. A)
Around the aorta (Ao) a population of QH1+cells was present: the majority is LEP 100+macrophages (arrowheads), but some QH1+/LEP1002CEC are
present (dotted areas, arrow). QH1+/LEP 1002CEC are observed in the circulation (red arrowhead). Bar: 40 mm B) Detection of an interstitial QH1+/
LEA2CEC (arrow) and of a QH1+/LEA+macrophage (arrowhead) in the brain mesenchyme (M), close to the neural epithelium (E). Bar: 20 mm. C) A
QH1+/LEA2CEC (arrow) reaches the endocardium. Note the presence of one quail QH1+/LEA+(white arrowhead) and two chick QH12/LEA+(red
arrowheads) macrophages. T: trabecula. Bar: 20 mm. D) Identification of three QH1+/vWF+CEC in the periaortic mesenchyme (arrows). Note the vWF+
endothelium of the aorta (Ao, arrowheads). Bar: 20 mm. E–H) Four examples of vessel-integrated (*) QH1+/Sambucus nigra+quail CEC (arrows) in the
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Yolk sac-derived CEC are mobilized during wound
We next tested if yolk sac derived CEC could participate to
repair processes such as wound healing. An incision was made in a
wing of E5.5 chimeras (Fig. 4A) and the embryos (n=17) were
sacrificed 3–8 hours later. Nineteen chimeras concerned chick
embryos grafted on quail yolk sacs, one experiment was performed
in the reverse combination. Histological observations showed that
the healing process took place rapidly as the wound depth was
dramatically reduced between 3 and 8 hours (Fig. 4B, C). An
important contingent of circulating cells invaded the wound cavity
as soon as 3hours after the operation (Fig. 4B, D). These cells were
a mix of QH12chick cells and QH1+quail cells (Fig. 4D). The
major part of quail cells was QH1+/LEP100+macrophages
(Fig. 4E) but QH1+/LEP1002cells were observed (Fig. 4E): as the
yolk sac is known to produce erythrocytes and macrophages,
QH1+/LEP1002cells present in the wound cavity could be CEC
since QH1 does not recognize erythrocytes. The invading QH1+
Figure 3. Distribution of chick yolk sac-born CEC in E15 quail chimeras. A–D and F) bone marrow cultures. A) Aspect of a quail bone marrow
culture after one day; Adipocytes with oil vacuoles (arrow), fibroblasts with filopodia (black arrowhead) and nucleated erythrocytes (red arrowhead)
are easily identified. The round cells present in the culture are HC. Bar: 40 mm. B) 8F3/vWF double staining identifies chick CEC (arrow) among 8F3+/
vWF2chick cells (arrowheads). Bar: 20 mm. C) The QH1/vWF double staining shows a chick CEC (arrow) among quail EC (white arrowheads) and HC
(green arrowheads). Bar: 20 mm. D) A QH12/vWF+/8F3+CEC is detected in a bone marrow smear. Bar: 10 mm. E) Table 3: Percentage of chick cells
invading the bone marrow in the oldest yolk sac chimeras. The number of 8F3/Hoechst cells is compared with the total cell population in cultures. F)
Presence of QH1+HSC among nucleated erythrocytes in a blood smear. Bar: 20 mm. G) Identification of a group of QH12/vWF+/8F3+CEC in a blood
smear. Bar: 20 mm. H) Triple staining on a transverse section through the intestine illustrating the participation of chick QH12/Sambucus+/LEP1002
CEC (arrows) to the vascular plexus of smooth muscle layers (M), beneath a villus (V). The quail host vessels are QH1+/Sambucus+/LEP1002and
appear purple (arrowheads). Bar: 40 mm. I) On a pia mater section, an interstitial QH12/Sambucus+/LEP1002CEC (arrow) is identified among QH1+
HC (arrowheads). Bar: 20 mm. J) An interstitial QH12/Sambucus+/LEP1002CEC (arrow) on a CAM section close to a QH1+vessel (V) and to QH1+HC
(arrowheads). Note that the endodermal layer (E) of the CAM is underlined by Sambucus and QH1 and appears purple. Bar: 20 mm.
wing (E), the dermis (F), at the vicinity of the dorsal root ganglia (G, DRG) and in the ventral vascular plexus of the neural tube (H). Bar: 20 mm. I) Table
1: Quantification of quail yolk sac derived CEC in the chick embryo. Counting of QH1+CEC in the embryo shows that the total number of yolk-sac
derived CEC (clear gray) increases between 1 and 4 days after grafting. However, as the embryo size expands during the same period, the CEC
concentration per mm3of embryonic tissue (dark gray) decreases. J) Table 2: Percentage of CEC that reached host endothelium.
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cells were distributed throughout the wounded wing, even in areas
distant from the wound site (Fig. 4B, C). In the wing mesenchyme,
part of the invading quail cells were not macrophages (Fig. 4F) and
did not belong to the hematopoietic lineage (Fig. 4G). Further-
more, QH1/vWF and QH1/Sambucus nigra double stainings
permitted to clearly identify CEC among the invading population
(Fig. 4H, I). These CEC were interstitially located or integrated in
capillary endothelia (Fig. 4H, I) in equivalent proportion.
Counting of QH1+CEC showed a statistically significant higher
concentration in wounded wings compared with the number of
QH1+CEC in contralateral wings (Fig. 4J). Interestingly, this
increase of QH1+CEC in wounded limbs was effective as soon as
3h after the surgery (Fig. 4J), suggesting that the mobilization of
QH1+CEC during wound healing was a rapid process.
The embryo proper does not produce CEC
To determine if the embryo proper was able to produce CEC,
we constructed yolk sac chimeras in which a quail embryo was
grafted on a chick yolk sac in a chick egg. In these conditions, the
capacity of the embryo to produce CEC could be attested by the
presence or the absence of QH1+EC in the chick yolk sac.
Twenty-seven yolk sac chimeras were constructed. Their survival
was44% afteroneday,44%aftertwodays, 33%afterfourdaysand
50% after five days. The oldest quail embryos we sacrificed reached
6.5–7 days of development (stage 23 Zacchei, 1961 ; Fig. 1D).
Serial sections of 11 chimeras were prepared, 3 after one day, 3
after two days, 3 after four days and 2 after five days (Fig. 1D).
Histological observations of chimeras, one day after the graft,
showed that a small rim of quail yolk sac was always grafted
Figure 4. CEC mobilization during wound healing. A) An E5.5 chick embryo (C) developed on a quail yolk sac (QYS). A wound is made in the
right wing bud (arrow), which is accessed through a hole in the amnion (*). Bar: 1.2 mm. B, C) Longitudinal sections of wounded wings. Three hours
after the operation (B), at the level of the brachial artery (BA), the wound (W) is invaded by an important contingent of chick (blue) and QH1+quail
cells (brown). After 8h (C), the rapid healing of the wound (W) is obvious. Note that numerous QH1+cells (arrowheads) are present in the wing
mesenchyme (B, C). Bar: 85 mm. D) High magnification of the mixed population of brown quail cells (Q) and blue chick cells (C). Bar: 20 mm. E) QH1/
LEP 100 double staining permits to discriminate between QH1+/LEP 100+macrophages (arrowhead) and QH1+/LEP 1002cells (arrow) in the wound.
Bar: 40 mm. F–H) Identification of QH1+cells in the mesenchyme of wounded wings. F) QH1+cells are not macrophages (arrows). Bar: 20 mm. G) A
QH1+/BEN+HC (arrowhead) is identified close to a non hematopoietic QH1+/BEN2cell (arrow). H) In the vicinity of a QH12/vWF+chick vessel (V),
QH1+/vWF+EC (arrows) are present with QH1+/vWF2HC (arrowhead). I) In the reverse combination, quail embryo grafted on chick yolk sac, a section
of QH12/Sambucus+chick vessel (arrow) present among the QH1+/Sambucus+quail vascular plexus (arrowheads). Bar: 40 mm in F, 30 mm in G–I. J)
Table 4: Mobilization of yolk sac derived CEC during wound healing. A wound on wings induces a statistically significant mobilization of CEC (dark
gray) by comparison with CEC concentration in contralateral limbs (clear gray). This mobilization is effective as soon as 3h after wounding (right part
of the table).
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PLoS ONE | www.plosone.org7 October 2011 | Volume 6 | Issue 10 | e25889
Figure 5. Absence of embryo-derived CEC. A) Transverse section through the trunk of a yolk sac chimera (quail embryo/chick yolk sac) one day
after the operation. On the right, the grafted quail territory in which all the vessels are QH1+(green) includes the embryonic area (QEA) and a part of
the quail yolk sac (QYS). The distinction between the two regions is histologically possible comparing their respective unspecific LEA-stained
endoderm: the endoderm of the embryonic area (white arrowheads) is thinner than the yolk sac endoderm (red arrowhead). On both sides, the quail
tissues are well associated with the chick yolk sac (CYS). The blue arrowheads point to three isolated QH1+cells migrating in chick yolk sac vessels.
Coe: coelom; NT: neural tube; S: somite; *: aorta. Bar: 110 mm. B) A QH1+/LEA2CEC (arrow) interstitially migrates at the surface of the chick yolk sac
endoderm (*). C) A QH1+CEC (arrow) reaches the endothelium of a yolk sac vessel (V). Bar: 20 mm in B and C. D) Table 5: Comparison of CEC number
in quail yolk sac-chick embryo chimeras (dark gray) and chick yolk sac quail embryo chimeras (clear gray). This histogram shows that in the
combination chick yolk sac/quail embryo this number is constant and residual (clear gray) by comparison with CEC concentration in the opposite
combination (dark gray). E) Scheme of the in vitro quail-chick chimera procedure: on a semi-solid medium (orange) a quail embryonic territory,
completely isolated from its yolk sac, and a whole chick blastoderm (embryonic + yolk sac areas) are placed side by side to permit the establishment
of vascular connections. F–I) QH1 immunohistochemistry on transverse sections of in vitro chick-quail chimeras 1.5 day after the operation: F) The
quail embryo is viable as shown by 1) chick blood cells (red arrowhead) in the aortae (Ao), which attested that a circulation is established with the
Origin of Circulating Endothelial Cells
PLoS ONE | www.plosone.org8 October 2011 | Volume 6 | Issue 10 | e25889
together with the quail embryo, at least on one side (Fig. 5A). The
contaminating quail yolk sac territory was easily identified on
sections by the aspect of the unspecific LEA-staining of the
endoderm, which appeared thicker than the endoderm of the
embryonic region (Fig. 5A) as previously described . If this
contamination had no importance when the yolk sac potentiality
was studied, it had to be taken into account when the embryo
capacity was studied. Nevertheless, due to the little contaminating
yolk sac we observed, we decided to analyze the chimeras.
The vascularization of grafted quail embryos appeared normal
as attested by continuous QH1 immunolabeling of the aorta, the
splanchnopleural and somatopleural vessels (Fig. 5A) and the
endocardium (not shown). Transverse sections of chick yolk sacs
showed the presence of round QH1+HC in the lumen of the
vitelline vessels (Fig. 5A). A few QH1+/LEA2CEC were
interstitially located (Fig. 5B) or integrated in endothelia
(Fig. 5C). Calculating the concentration of QH1+/LEA2CEC
in chick yolk sacs, we found a very low number by comparison
with number of CEC found in the reverse combination (Fig. 5D)
and this concentration did not vary during all stages examined
(Fig. 5D). In these conditions, we could not exclude that the
QH1+/LEA2CEC observed in chick yolk sacs came from the
piece of contaminating quail yolk sac grafted with the quail
embryo. Indeed, the less contaminated yolk sac we grafted, the less
QH1+CEC we observed in chick yolk sacs, suggesting that these
cells came from the contaminating quail yolk sac and not from the
quail embryo proper.
Attempts to perform the same experimental protocol by grafting
quail embryos totally devoid of yolk sac tissue were never
successful, as the suture between quail and chick territories was
impossible to maintain due to the high tension between tissues at
this level (not shown).
To palliate this technical difficulty, we created in vitro chimeras
consisting of a chick embryo with its yolk sac (10–13 s.s.) placed
next to a quail embryo (9–12 s.s.) without its yolk sac, on a
semisolid medium (Fig. 5E). In these conditions, the suture
between the edges of tissues was correctly maintained, the tension
forces being less important on the semisolid medium. We
previously showed that in vitro chimeras could survive, could be
studied during two days and that quail CEC could colonize chick
territories . On 10 associations, 4 were analyzed: the quail and
chick embryos respectively reached 15–20 s.s. and 15–23 s.s.. As
quail embryos were unable to produce primitive HC due to the
absence of their yolk sac, their survival attested the presence of
vascular anastomoses with the chick territory allowing to chick HC
to feed the quail embryo: the histology confirmed the presence of
chick primitive HC in the lumen of the quail vessels (Fig. 5F, G).
Morphologically, quail embryos developed a normal vascular tree
(Fig. 5F) with functional heart and aorta. At the level of the quail-
chick junction, vascular connections were identified between quail
and chick vessels (Fig. 5G) and some QH1+EC could interstitially
migrate in the endothelium of proximal chick yolk sac vessels
(Fig. 5G). Analysis of the distribution of QH1+cells in chick
embryos showed absence of quail CEC in all tissues and
endothelia (Fig. 5H, I). Thus, using in vitro chimeras to study
the ability of the embryonic territory to generate CEC, we show
that the embryo per se is unable to produce these cells.
Is the yolk sac the only appendage producing CEC?
It was previously shown that the allantois was able to produce
CEC colonizing the bone marrow when ectopically grafted in the
coelom , but it remained to be determined if it had it the same
capacities in vivo. To resolve this point, we created, in chick eggs,
half embryo chimeras before the onset of circulation (13s.s.). This
surgical technique permitted to discover that HSC destined to
colonize intraembryonic organs arise in the whole embryo exept
the prospective head-neck region .
In these chimeras, the posterior part of a quail embryo, behind
the last formed somites, replaced its chick counterpart (Fig. 6A).
This surgery produced chimeras with a quail allantois developing
in the hind gut region. The resulting chimeras (5/37, Fig. 6B) were
autopsied 4 days after the operation at E5.5–6 (stages 27–28HH),
2.5 days after the establishment of vascular connections between
the embryo and the allantois (St19HH, ). The chimeras
developed correctly and the dorsal limit between chick and quail
territories was identified by a dilatation at the level where neural
tubes fused, sometimes involving the presence two tubes side by
side (Fig. S3A). As the grafts were performed before blood
circulation, the tissues developed from the quail territory included
the allantois (Fig. S3B), the wing (Fig. S3C), limb and tail buds, the
body wall and the dorsal structures such as the neural tube
(Fig. 6C). In these territories the vascular plexus was QH1+
(Fig. 6C, S3A–C). Interestingly, the viscera came from the chick
(Fig. 6C). These observations confirmed pioneer fate map
experiments showing that, until the level of the 15thsomite, the
lateral endomesoderm participates to the formation of the
digestive tract, but not to the hind gut . Furthermore, as
shown previously, the vascular plexus of viscera was chick and
QH12([41,42]; Fig. 6C). Heart and lungs (Fig. 6C) had also a
chick origin. Rostrally to the wing level, the tissues in the head
were chick (Fig. 6D). The vascular plexus became QH12/
Sambucus nigra+(Fig. 6D) and in the territories which were at the
boundary between chick and quail, chick EC progressively
replaced the QH1+endothelial plexus as in the aortic endothelium
(Fig. 6C). Finally, the yolk sac was chick and vascularized by
QH12EC (Fig. S3D).
Concerning CEC, as we found that the embryonic territory was
unable to produce these cells (see above), the identification of
QH1+CEC in chick tissues would mean that these cells came from
the allantois. A counting of CEC showed a concentration of 2–3
QH1+CEC/mm3(Fig. 6E). In the chick head we could detect
QH1+/vWF+, QH1+/Sambucus nigra+or QH1+/Sambucus+/
BEN2CEC, integrated in endothelia (Fig. 6F) or interstitially
located (Fig. 6G–K).
In these chimeras as the limit between chick and quail territories
was located rostrally to the wing level, all definitive HSC that arose
in the truncal ventral aortic region were quail, thus QH1+(Fig. 6C,
D) and were found in chick territories (Fig. S3E, F).
In conclusion, using half embryo chimeras, we showed that the
allantois produced CEC in situ and may generate QH1+EC found
chick territory, and 2) the presence of QH1+vessels in the somatopleural (arrow) and splanchnopleural mesoderms (arrowheads). Coe: coelom; NT:
neural tube; N: notochord; S: somite; *: cardinal vein. Bar: 50 mm. G) Region of contact between the two embryos: the broken line delimits the quail
territory on the left, with QH1+endothelia (*), and the chick territory on the right, with QH12endothelium: note the presence of two QH1+EC which
interstitially migrate in the proximal part of the chick vessel (arrowheads). Bar: 50 mm. H, I) Absence of QH1+cells illustrated on two different chick
embryos: H) in the hindbrain; I) in the umbilical region. Ao: aorta; CV: cardinal vein; N: notochord; NT: neural tube; U: umbilic; *: vessels. Bar: 90 mm in
Origin of Circulating Endothelial Cells
PLoS ONE | www.plosone.org9 October 2011 | Volume 6 | Issue 10 | e25889
Origin of Circulating Endothelial Cells
PLoS ONE | www.plosone.org10 October 2011 | Volume 6 | Issue 10 | e25889
in the bone marrow of older yolk sac chimeras in addition to the
In this study, we use yolk sac and half embryo chimeras to
demonstrate that CEC are generated in extraembryonic append-
ages, the yolk sac and the allantois, and travel through the
circulation to reach embryonic vessels in most organs as well as the
bone marrow. The density of CEC in these chimeras was
measured by counting the number of quail yolk sac derived
QH1+CEC per mm3of embryonic tissue (Fig. 2I, 6E). We have
previously demonstrated the existence of embryonic CEC using
quail-chick parabiosis, where two embryos are grown inside one
eggshell, allowing fusion of their choriallantois membranes and
blood circulation from one embryo to another . CEC number
in yolk sac and half embryo chimeras is equivalent to that
previously observed in quail-chick parabioses prior to bone
marrow formation, indicating that all CEC are generated in the
yolk sac and the allantois. We previously showed that in older
embryos only 5% of CEC were found in endothelia ; active
angiogenesis early in development apparently requires larger
numbers of CEC integrating into vessels.
Using yolk sac chimeras, Dieterlen-Lie `vre and Martin 
discovered that definitive HSC are not generated in the yolk sac,
but in the embryo proper. These findings have since been
confirmed in mice and humans [22,43]. The yolk sac produces a
transient wave of HC, mostly erythrocytes and macrophages,
which are later replaced by a new population of HSC born in the
para-aortic splanchnopleura and the ventral wall of the aorta .
Some of the yolk sac macrophages invade the embryonic central
nervous system and differentiate to microglia [44,45]. Histological
observations have suggested that all microglial cells derive from
yolk sac macrophages [44,46]. Recently, this has been confirmed
using lineage-tracing in mice . Taken together with the
findings reported here, these observations suggest that the yolk sac,
a transient extra-embryonic appendage, gives rise to two cell types
persisting in adult, which are microglial cells and CEC.
Constructions of yolk sac chimeras where a quail embryo was
grafted onto a chick yolk sac showed that the embryo proper is
unable to give rise to CEC. These experiments were particularly
challenging technically, as the quail embryo was hard to isolate
completely from yolk sac territory and in vitro chimeras had to be
generated. However, in spite of the technical difficulties, definitive
quail HSC and endothelium were readily seen in these chimeras.
Moreover, quail-derived CEC were only found in the chick yolk
sac when a piece of contaminating yolk sac had been grafted, and
totally absent in the in vitro associations. As quail-chick parabiosis
experiments had shown that the density of CEC in the yolk sac
and intra-embryonic tissue was similar , these results lead us to
conclude that the embryo proper cannot give rise to CEC.
Previous studies using quail-chick chimeras had suggested that
the allantois, another extra-embryonic appendage, could produce
CEC able to colonize the bone marrow after heterotopic grafting
into the coelom . Both the yolk sac and the allantois are purely
splanchnopleural appendages, i.e., formed from endoderm and
splanchnopleural mesoderm, and have hemangiopoietic capacity,
i.e., they can give rise to EC and HC. To test whether the allantois
preserves its hemangiopoietic capacity in situ, we constructed half
embryo chimeras and demonstrated that the allantois is able to
give rise to CEC. As intraembryonic splanchnopleural mesoderm
can produce EC and HSC, but not CEC, these data suggest that
instructive cues from extraembryonic endoderm might trigger
CEC formation as previously shown during hematopoietic
In mice, the allantois is very rudimentary and the placenta,
which has hemangiopoietic capacity, replaces its function .
Whether CEC are generated in extraembryonic territory,
including yolk sac and placenta in mice remains to be
experimentally addressed. As placental tissue is accessible, it
would be an attractive source of CEC in addition to HSC .
Our results indicate that CEC are generated in the yolk sac and
the allantois, suggesting that they are originating from few sites of
production and that they acquire diversity during later embryonic
stages, perhaps after homing to the bone marrow. The bone
marrow provides a suitable environment for the multiplication of
CEC, as CEC density in embryonic tissues increases significantly
after bone marrow arises in quail-chick parabiosis . Factor(s)
inducing CEC multiplication in the marrow and inhibiting CEC
multiplication in tissues remain to be identified. Preliminary data
suggest that VEGF could play a role in the migration of yolk sac-
derived CEC (LP and AE, unpublished data), but additional work
is required to identify mechanisms controlling embryonic CEC
Although they are rare in embryonic tissues, CEC can be
mobilized during acute angiogenesis process, i.e., wound healing.
Previous experiments using parabiosis had demonstrated that
CEC numbers increase significantly after induction of neovascu-
larization by wounding or by grafting of an organ on the CAM,
which is then vascularized . CEC also participated in small
numbers to vascularization of tumors grafted onto the chick CAM
of parabioses . In all cases, they were found in part outside of
vessels, suggesting that rather than contributing directly to
neovessel formation, they stimulate angiogenesis by release of
soluble factors [13–15]. This simple model thus reproduces results
obtained in mice and reveals an indirect contribution of CEC to
marrow. A) Two 8F3+chick cells (arrow) observed with
Identification of chick HC in the bone
Figure 6. half embryo chimeras. A) A 9 s.s. half embryo chimera just after the operation: the dotted region corresponds to the grafted quail
territory (Q) caudally to the last formed somite. The rest of the chimera is chick (C). Note that the quail and chick neural tubes are aligned (arrow). Bar:
1 mm. B) A half embryo chimera 4 days after the operation: the embryo is well developed with its wing (*) and limb (u) buds; the dotted line shows
the limit between chick (C) and quail (Q) territories. The quail allantois (QAL) is present above the head. Bar: 2 mm. C) A cross section at the truncal
level showing the chick intrinsic QH12/Sambucus+vascularization in the gut (G) and the lungs (Lu). Dorsally, the vascular tree is quail, QH1+/
Sambucus+, in particular around the neural tube (NT), the cardinal veins (*) and the umbilical vein (u). Note that the aortic endothelium (Ao) is
chimeric with a part of chick EC located ventrally (arrowhead). Sambucus also labels basement membrane of epithelia in gut, lung and liver (L). N:
notochord; M: mesonephros. D) A cross section in the diencephalon. All the vascular network is chick, i. e., QH12/Sambucus+in particular around the
right eye (E), the diencephalic vesicle (Di) and the right jugular vein (JV). The green dots are QH1+HC. Ph: Pharynx; T: tongue. Bar: 80 mm in C, D. E)
Table 6: Counting of quail CEC identified in chick territories of half embryo chimeras. Less than 3 QH1+CEC/mm3are present. F) Close to the
diencephalic epithelium (E), a QH1+/Sambucus+CEC (arrow) is integrated in a chick QH12/Sambucus+vessel (V). G) Identification of a QH1+/vWF+CEC
(arrow) interstitially located in the diencephalic mesenchyme. Bar: 20 mm in F, G. H–K) QH1/Sambucus/BEN triple staining confirms the endothelial
nature of a quail purple CEC (arrowhead) in the diencephalic mesenchyme. Bar: 40 mm in H–K.
Origin of Circulating Endothelial Cells
PLoS ONE | www.plosone.org 11October 2011 | Volume 6 | Issue 10 | e25889
QH1+cells among a majority of QH12/8F32quail population.
Bar: 20 mm. B) Two 8F3+/LEA2chick cells (arrows) and two
8F32/LEA+quail macrophages (arrowheads) are visible in this
field. C) QH/BEN staining identifies double stained HC and
one QH1+/BEN2cell (arrowhead). Bar: 20 mm. Bar: 20 mm in
phages in E15 quail chimeras. A) In toto double immuno-
staining showing 8F3+cells (green) invading the QH1+vascular
plexus of the pia mater. Bar: 20 mm. B) High magnification of a
8F3+cell showing a thin filopodial extension (arrow) towards the
vascular plexus. Bar: 7 mm. C) Triple staining on a section
through the pia mater identifying a chick QH12/Sambucus+/
LEP100+macrophage (arrow) among QH1+HC (arrowheads).
Bar: 20 mm. D) In toto double immunostaining in the skin
showing a chick cell (arrowhead) present among QH1+cells. Bar:
20 mm. E) Skin section with a triple staining identifying a chick
QH12/Sambucus+/LEP100+macrophage (arrow) among QH1+
HC (arrowheads). Bar: 20 mm. F) Transverse section through the
heart with two chick QH12/Sambucus+/LEP100+macrophages
(arrows) close to a QH1+coronary vessel (V). Bar: 20 mm. G–I) In
toto QH1/8F3 double staining in large vessels does not identify
8F3+chick cells in the quail aortic endothelium (G), the quail
aortic vasa vasorum (H) and the quail jugular vein endothelium (I).
Distribution of chick yolk sac-born macro-
Note the presence of QH1+HC at the aortic luminal surface (G,
orange dots). Bar: 30 mm in G and I, 10 mm in H.
illustrates the junction region between quail and chick territories
where quail (QNT) and chick (CNT) neural tubes overlap. In the
quail region QH1+vessels are present. In the chick territory, a part
of the neural tube is vascularized by quail EC that have migrated
interstitially. The QH1+dots on the left are quail HC. B) Section
of the quail allantois vascularized by QH1+vessels. C) QH1+
vessels present in the quail wing bud. D) Section of the chick yolk
sac with QH12vessels in which QH1+HC (arrows) are observed.
E) Presence of a quail QH1+/BEN+
rhombencephalic mesenchyme together with a chick QH12/
BEN+HC (arrowhead), probably a macrophage. Note that BEN
stains the neuronal plexus (*) in the epithelium (E). F) LEP100+
macrophages detection in the diencephalic mesenchyme. One
double stained QH1+/ LEP 100+quail macrophage (arrow) is seen
among chick ones (arrowheads). Bar: 40 mm in A–F.
Half embryo chimeras. A) This cross section
HC (arrow) in the
Conceived and designed the experiments: LP AE. Performed the
experiments: LP. Analyzed the data: LP AE. Contributed reagents/
materials/analysis tools: LP AE. Wrote the paper: LP AE.
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