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Vasculogenesis of the embryonic heartOrigin of blood island-like structures.

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THE ANATOMICAL RECORD PART A 288A:223–232 (2006)
Vasculogenesis of the Embryonic
Heart: Origin of Blood Island-Like
Structures
ANNA RATAJSKA,1* ELŻBIETA CZARNOWSKA,2
AGNIESZKA KOŁODZIŃSKA,1 WOJCIECH KLUZEK,1 AND
WOJCIECH LEŚNIAK1
1
Department of Pathological Anatomy, Medical University of Warsaw,
Warsaw, Poland
2
Department of Pathology, Children’s Memorial Health Institute, Warsaw, Poland
ABSTRACT
The earliest vascular structures (blood island-like) in the embryonic heart are clusters of
angioblasts and nucleated red blood cells (NRBCs), which differentiate into endothelial cells
and erythrocytes, respectively. Our purpose was to define the area and chronology of NRBC
appearance in the mouse embryonic heart at the stages before a patency between coronary
vessels and peripheral circulation is established (10.5–13.5 dpc). Before and at the onset of
vascularization, NBCs were not present within the proepicardium; however, Ter/119⫹ differentiating erythroblasts and single scattered CD45⫹ were found in the heart beginning
from 10.5 dpc. The Ter/119⫹ cells were in close apposition to angioblasts (PECAM1⫹) and
were recognized as components of blood island-like structures or vascular vesicles in transmission electron microscope and were located mostly in the subepicardium. Some of the
NRBCs were not accompanied by angioblasts and located close to the endocardial endothelium or at the border of the endocardial endothelium or in the subepicardium. These erythroblasts were beginning to assemble with angioblasts. CD34⫹ NBCs as well as progenitor
cells of erythroid lineage were not detected in the heart at these stages of development. The
state of differentiation of NRBCs of blood islands was similar/the same as the morphology of
circulating blood cells at the respective stages of embryo development. The presence of
mature NRBCs in the subendocardial area and lack of progenitor cells of erythroid lineage
within the heart indicate that erythroid commitment occurs outside the heart. We suggest
that NRBCs enter the heart from the blood stream at 10.5–12 dpc independently from
angioblasts. © 2006 Wiley-Liss, Inc.
Key words: vasculogenesis; angiogenesis; hematopoiesis; murine heart; proepicardium; nucleated red blood cells
Vessel development within the embryonic heart occurs
via two processes: vasculogenesis and angiogenesis (Rongish et al., 1994; Risau, 1995, 1997). Vasculogenesis is the
formation in situ of coronary vessels from endothelial cell
progenitors (angioblasts) or angioblast migration to areas
of vessel formation and their subsequent differentiation
into vascular channels. Angiogenesis is the development
of vessels from preexisting ones by capillary sprouting,
intussusceptive growth, and remodeling (Risau, 1997;
Ratajska et al., 2003). The first morphological signs of
vasculogenesis within an embryo are “blood islands,”
which consist of erythroblasts and premature endothelial
cells (angioblasts). Blood islands are encountered within
the yolk sac of 7.5–13 dpc embryos. The first sings of
vasculogenesis in heart development are blood island-like
structures. This term was introduced due to the struc©
2006 WILEY-LISS, INC.
tures’ morphological resemblance to the blood island of the
yolk sac (Rongish et al., 1994). These structures assemble
Grant sponsor: Polish State Committee for Scientific Research;
Grant sponsor: Medical University of Warsaw; Grant number: 6
P05A 025 20 2P05A 111 28.
*Correspondence to: Anna Ratajska, Department of Pathological Anatomy, Medical University of Warsaw, Chałubińskiego 5,
02-004 Warsaw, Poland. Fax: 48-22-6299892.
E-mail: arataj@ib.amwaw.edu.pl
Received 14 November 2005; Accepted 26 November 2005
DOI 10.1002/ar.a.20311
Published online 6 February 2006 in Wiley InterScience
(www.interscience.wiley.com).
224
RATAJSKA ET AL.
in clusters prior to their presumed coalescence and differentiation into vascular channels (Tomanek et al., 1996).
Blood island-like structures are located within the myocardial wall and the subepicardium of 10 and 11 dpc mice
(Virágh and Challice, 1981). They are encountered within
13 dpc rat hearts (Rongish et al., 1994; Ratajska and
Fiejka, 1999). They are also found in the embryonic heart
in later stages of development (until full term). Little is
known about the derivation of the blood cell component of
the blood island-like structures and their relationship to
hematopoietic differentiation stages.
Heart vascularization immediately follows the epicardial expansion. Hearts devoid of the epicardium do not
develop vascular channels (Kwee et al., 1995). The epicardium derives from an extracardiac source (Kurkiewicz,
1909): proepicardial villi, which form on the ventral surface of the sinus venous horns as cellular protrusions
growing toward the pericardial cavity and reaching the
opposite surface of the looping heart (dorsal atrioventricular sulcus) (Ho and Shimada, 1978; Virágh and Challice,
1981; Kuhn and Liebherr, 1988; Hiruma and Hirakow,
1989; Männer, 1992; Virágh et al., 1993; Männer et al.,
2001). The proepicardium subsequently spreads over the
surface of bare myocardium, giving rise to the epicardium
and the subepicardial mesenchyme. Some cells of the epicardial mesothelium possess the ability to migrate to the
subepicardial space undergoing epicardial-mesenchymal
transformation. These mesenchymal cells are considered
to be a source of smooth muscle cells that make up the
tunica media of coronary vasculature and interstitial fibroblasts as well as fibroblasts of the adventitia (Mikawa
and Gourdie, 1996; Dettman et al., 1998; Gittenberger-de
Groot et al., 1998; Vrancken Peeters et al., 1999; Wada et
al., 2003). Subepicardial space, particularly at the atrioventricular and interventricular sulcuses, is filled with
blood cells, angioblasts, mesenchymal cells, and extracellular matrix material (Virágh et al., 1993; Kálmán et al.,
1995).
The origin of coronary vascular endothelial cells is still
controversial. One theory claims that endothelial cells
derive from liver primordium/sinus venosus region and
are transported via proepicardium to the heart’s surface
(Poelmann et al., 1993, 2002). According to the other theory, endothelial cells derive from mesenchymal cells after
epithelial-mesenchymal transformation of the epicardial
mesothelium (Mikawa and Gourdie 1996; Muñoz-Chápuli
et al., 1999, 2002; Pérez-Pomares et al., 2002). Although
both theories have accumulated some experimental evidence, the derivation of endothelial cells within the embryonic heart is ambiguous. There is also a possibility that
coronary endothelial cells are of dual origin. Regardless of
their origin, they arrive to the heart from the epicardial
surface. Close association of the nucleated red blood cells
(NRBCs) with primordial endothelial cells at early stages
of vasculogenesis might indicate that either both kinds of
cells derive from a common precursor, or blood cells derive
from migrating angioblasts (Morabito et al., 2002). The
existence of hemangioblast as the common progenitor of
the angioblast and blood cell has been suggested before
(Pardanaud et al., 1989). The current evidence for this
opinion comes from common molecular markers in cells of
endothelial/angioblastic and hematopoietic potential (Millauer et al., 1993; Lin et al., 1995; Shalaby et al., 1995;
Young et al., 1995; Eichmann et al., 1997). In addition, the
areas of embryos where hematoblasts appear are strictly
related to hematopoietic and vasculogenic events (yolk
sac) (Haar and Ackerman, 1971; Tavassoli, 1991). The
issue of the relationship between hematopoietic cells and
vasculogenesis in embryonic heart development has been
raised in a current study by Kattan et al. (2004). We
wanted to add some relevant data to this topic.
Our purpose was to address derivation of blood cells
that are constituents of blood islands of the embryonic
heart at the stages before a connection between coronary
vessels and the peripheral circulation is established. We
wanted also to study a chronology of the appearance of
NRBCs in the embryonic hearts and to characterize the
relationship between vascular structure appearance and
hematopoietic stage of NRBC. Proepicardium as a possible
source of NRBC was also explored. All these studies were
performed on mouse embryos at the stages since 9.5
through 13.5 dpc.
Although the embryonic hematopoiesis has been the
topic of many experimental papers and recent reviews
(Dzierzak et al., 1998; Dzierzak, 2002, 2003), the issue of
the embryonic heart as a hematopoietic organ has been
addressed (Virágh et al., 1990, 1993; Kálmán et al., 1995)
but has not been proven so far. Thus, red blood cells within
early vascular structures of the embryonic heart may derive from common hemangioblastic precursors of endothelial cell and blood cell lineage or from independent hematoblastic or endothelial cell lines. They may also enter the
heart directly from circulation at the established stage of
erythroid differentiation.
MATERIALS AND METHODS
All procedures were performed according to requirements of the Ethical Animal Care Committee of Poland
(which is also in accordance with the National Institutes
of Health Guidelines for the Care and Use of Laboratory
Animals).
Animals (pregnant mice, 9, 9.5, 10, 10.5, 11, 11.5, 12,
12.5, 13.0 dpc) were sacrificed under anesthesia (narcotan
and chloral hydride 100 mg/kg b./w. i.p.); fetuses were
removed, decapitated, and fixed immediately in 2.5% glutaraldehyde/2% paraformaldehyde solution in 0.1 M cacodylate buffer, pH 7.6, for 3 hr. Subsequently, tissue was
rinsed in 0.1 M cacodylate buffer, osmicated in 1% OsO4
for 45 min, dehydrated in graded series of ethanol solutions, and embedded in Spurr resin. Embryos were oriented for sagittal sectioning. Semithin sections were cut
serially every 20 –30 ␮m and were stained with toluidine
blue for analysis under a light microscope.
Some embryos were fixed in zinc fixative to preserve
antigenic determinants of CD34 (Beckstead, 1994), or in a
buffered formalin (to preserve the antigenic determinant
of Ter/119 and CD45), dehydrated in increasing alcohol
concentrations, cleared in xylene, and embedded in paraffin. Other set of embryos were frozen immediately for
obtaining cryostat sections, suitable for anti-PECAM1
staining and some hematopoietic markers staining. At
least three embryos for each of the above-mentioned time
points were sacrificed and taken for analysis.
Immunohistochemistry
Paraffin sections were deparaffinized on a hot plate and
3 in changes of xylene and alcohol, 10 min each. For
detection of CD45 antigen sections were treated with Antigen Retrieval solution according to manufacturer re-
BLOOD ISLAND-LIKE STRUCTURES
225
quirements (Pharmingen). Sections were then incubated
with rat anti-CD34 (1:30; Pharmingen), anti-CD45 (1:30;
Pharmingen), or anti-Ter/119 (1:30; Pharmingen) antibodies for 60 min in a humid chamber or overnight at 4°C,
washed with TBS, and subsequently treated for 30 min
with antirat IgG-biotin conjugate (Pharmingen). After
washing in TBS, the sections were stained with streptavidin-peroxidase complex (Dako) and the color reaction
was developed with DAB (Sigma). For double labeling,
frozen sections were cut and stained with anti-Ter/119 (or
anti-CD34) antibodies and the same sections were stained
with goat anti-PECAM1 antibodies, (Santa Cruz Biotechnology), rinsed as above, and incubated with antigoat alkaline phosphatase complex (Sigma) and antirat-IgG-biotin conjugate. After rinsing with TBS, streptavidinperoxidase complex was applied on sections. The color
reaction was developed for horseradish peroxidase with
(DAB) substrate and subsequently for alkaline phosphatase with NBT/BCIP substrate (Dako) or Fast Red substrate (Dako). The sections were finally stained with hematoxylin-eosin and mounted in glycerogel (Keiser).
Ultrastructural Analysis
Semithin sections stained with toluidine blue were analyzed under a light microscope. Selected samples chosen
from semithin sections were cut for ultrathin sections for
copper grids, counterstained with lead nitrate and uranium acetate according to Reynolds (1963), and examined
in a transmission electron microscope (TEM).
RESULTS
To explore possible sources of NRBCs within the early
vascular structures of mouse hearts, we examined initially the proepicardium (9.5–10 dpc) and the avascular
heart (since 10.5 dpc) for the presence of hematopoietic
progenitor cells.
Proepicardium
On 9 –9.5 dpc, the proepicardium consisted of loosely
arranged mesenchymal cells (Fig. 1a) and of cells that
exhibited epithelial morphology (Fig. 1b). In TEM, mesenchymal cells developed long cytoplasmic processes of irregular shapes, which were connected with adjacent cells.
Mesenchymal cells contained rich Golgi complex and scattered filaments of an intermediate type. Epithelial cells of
proepicardium formed primitive intercellular junctions
(Fig. 1c). They formed blebs, which protruded toward the
pericardial cavity. From the tips of these protrusions,
vesicles were released to the pericardial cavity (Fig. 2a
and b). The vesicles consisted of one or several epithelial
cells. The blebs and vesicles were never positive with
anti-CD34 or anti-CD45 antibodies on serial sections (Fig.
2c), which indicates the absence of hematopoietic precursors and endothelial cells in the proepicardium (Wood et
al., 1997; Jaffredo et al., 1998). CD34⫹ endothelial cell
precursors were, however, detected in the vicinity of the
proepicardium within the septum transversum/primitive
liver. The highest number of vesicles within the pericardio/peritoneal cavity was detected on 9.5 dpc. After the
vesicles had reached the dorsal surface of the ventricles,
they flattened and spread, forming the cover of the heart.
The process of the epicardial cover development was accomplished on 11 dpc and after this stage the free-floating
vesicles were not encountered within the cavity. Occa-
Fig. 1. An embryo on 9.5 dpc. Ultrastructure of the proepicardium at
the early stage of its development. a: Loosely arranged mesenchymal
cells with long processes are the only constituents of the proepicardial
matrix. b: The proepicardial surface consisting of primitive epithelial
cells. c: Intercellular junction (arrow) between adjacent epithelial cells.
Magnifications: 5,000⫻ (a); 7,200⫻ (b); 21,000⫻ (c). Abbreviations for all
figures: a, atrium; an, angioblast; c, myocardial cell; e, erythroblast,
NRBC; en, endocardial endothelium; ep, epicardial epithelium; o, outflow tract; s, sinus venosus; v, ventricle.
226
RATAJSKA ET AL.
sional proepicardial vesicles were detected in close association with the heart’s surface of 11 dpc embryos.
In myocardium, neither nucleated blood cells nor their
precursors (CD34⫹ or CD45⫹) were detected at the stages
preceding heart vascularization (9.5–10 dpc), although endocardial endothelial cells expressed CD34 antigen (Wood
et al., 1997).
Myocardium and Subepicardium
Fig. 2. A 9.5 dpc mouse embryo. A sagittal section demonstrates the
proepicardium with cellular vesicles. a: A small magnification of the
embryo with an area marked in the rectangle enlarged in the consecutive
section. b: Higher magnification of the proepicardium. Vesicles are released from the tip of the proepicardium (arrow). Hematoxylin-eosin
staining. c: Anti-CD34 labeling of the embryo. The proepicardium does
not express CD34 activity (arrow). Magnifications: 80⫻ (a); 380⫻ (b);
200⫻ (c).
Beginning on 10.5 dpc, NRBCs were found in the wall of
embryonic heart in association (in apposition) with angioblasts. They appeared as clusters of closely apposed cells
or entrapped in loose assemblies within vascular vesicles.
The latter were located in the subepicardium within the
interventricular or atrioventricular sulcuses (Fig. 3). Clusters of NRBCs and angioblasts were recognized as blood
island-like structures in TEM. The NRBCs residing in
blood islands express Ter/119 antigen, whereas endothelial cells exhibited PECAM1 antigen. In addition to the
previous TEM studies (Rongish et al., 1994; Ratajska and
Fiejka, 1999), which claimed that red blood cells were
accompanied by angioblasts/endothelial cells, we demonstrated that some of the NRBCs were located in areas
where angioblasts were absent. These solitary (free) NRBCs were mostly positioned in the subendocardium. However, some were found also in the subepicardium. The
highest number of solitary NRBCs was demonstrated on
11.5–12 dpc. They were recognized on semithin sections
(Figs. 4 and 5a) by the use of immunohistochemical labeling with anti-Ter/119 antibodies (Fig. 5b– d) and in TEM
(Fig. 5e). They did not express CD34 antigen, indicating
that they did not belong to cells of early hematopoietic
lineage. Double labeling with anti-PECAM1 and anti-Ter/
119 antibodies allowed us to confirm that Ter⫹ cells resided subendocardially and were not encircled by PECAM⫹ angioblasts. However, they adjoined PECAM⫹
endocardial endothelium (confirmed by TEM study; Fig.
5e). Single cells exhibiting Ter/119 antigen were also
found to be located on the border of the endocardial endothelium, as if passing through it (Figs. 5d and 6). Some
subepicardially located NRBCs adjoining the epicardial
epithelium were also distinguished (Fig. 7). An ultrastructural study indicated that NRBCs moved to establish contact with angioblasts. Moving blood cells were recognized
morphologically by the presence of pseudopods (Fig. 8a).
Subsequently, NRBCs were being enveloped by angioblasts (Fig. 8). Pseudopods or long processes were also
visible during angioblast recruitment to blood islands
(Fig. 9).
In the subepicardium and myocardium of 11–13 dpc
hearts, most NRBCs were accompanied by angioblasts/
endothelial cells. This close vicinity of NRBCs and angioblasts represented blood island-like structures. Both circulating NRBCs and residing NRBCs (in blood islands) of
the same stage of differentiation consisted of a population
of cells that differed with respect to their maturation
stages. However, among residing NRBCs, the early erythroblastic progenitors were never encountered. Ter/119⫹
NRBCs residing in the heart belonged to late erythroblastic stages of differentiation (a halo around a nucleus indicated the commencement of the nucleus shedding; Fig.
10). There were some singly scattered CD45⫹ cells in
peripheral circulation and residing within the heart of
10.5–13 dpc embryos. Some residing NRBCs became enucleated by expulsion of the nucleus (not shown), whereas
BLOOD ISLAND-LIKE STRUCTURES
227
Fig. 3. An 11 dpc heart, semithin sections. a: Nucleated red blood
cells within the subepicardial vascular vesicles are visible (rectangles).
The vesicles are located within the interventricular (b) and atrioventricular
(c and d) sulcuses. d: Some nucleated red blood cells within the vascular
tube proliferate. Mitotic figures are marked with arrows. Magnifications:
50⫻ (a); 300⫻ (b– d). [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com].
blood cells circulating in the peripheral blood stream
maintained their nuclei longer (Fig. 10b; they lose their
nuclei beginning on 15 dpc). Enucleated RBCs coexisted
with nucleated ones in blood island-like structures beginning from 12 dpc. Thus, blood islands of the same stage of
heart development differentiate and mature at different
time points, not simultaneously.
be located on the border of the endocardial endothelium
(Figs. 5d, 6), as if pictured while moving across the endocardial endothelium. Although we have not studied the
movement of these cells, our indirect morphological observations strongly suggest that NRBCs move through myocardium to come in contact with angioblasts (presence of
cytoplasmic processes). They enter the heart at the stage
of development corresponding to the established erythroid
lineage, not at the stage of hematopoietic progenitor cells.
This was confirmed by their more mature morphology
(ultrastructure) as compared to the erythroblasts of yolk
sac blood islands (Haar and Ackerman 1971; Tavassoli,
1991) and their Ter/119 antigen expression, the latter
being specific to cells of erythroid/erythrocyte stage (Ikuta
et al., 1990). A similar observation, namely, that commitment to hematopoietic lineage precedes the formation of
blood island-like structures in the embryonic heart, has
been suggested in a previous study by Kattan et al. (2004).
Contrary to the authors’ finding, we have not observed
many CD45⫹ cells within the blood island structures and
vascular vesicles of the 10.5–13 dpc hearts. This discrepancy might be caused by species-specific differences in
CD45 antigen expression (quail versus mouse). NRBCs
encircled by endothelial cells/angioblasts are still able to
proliferate, which indicates that not all of these cells are
at the terminal stage of erythroid differentiation.
The proepicardium is generally known as the precursor
of the epicardial mesothelium, which, after mesenchymal
transformation, gives rise to cellular components of the
coronary vasculature (Mikawa and Gourdie, 1996; MuñozChápuli et al., 1996, 2002; Dettmann et al., 1998; Gittenberger-de Groot et al., 1998; Pérez-Pomares et al., 2002;
Poelmann et al., 2002). The proepicardium has also been
acknowledged as having a potential to deliver endothelial
DISCUSSION
Our study has provided several important observations
regarding red blood cell characteristics and chronology of
red blood cell occurrence in the blood island-like structures of the embryonic heart. One, at the onset of heart
vascularization, NRBCs are found in the heart either accompanied or not accompanied by angioblasts. Two, the
solitary (free) NRBC (i.e., not accompanied by angioblasts)
are found to be located mostly in the subendocardium, on
the border of endocardial endothelium, or within the subepicardium, whereas the ones accompanied by angioblasts/endothelial cells are found in myocardium and in
the subepicardium. Three, free NRBCs establish contact
with angioblasts. Four, premature erythroid cells and/or
hematopoietic stem cells were not encountered in 10.5–13
dpc hearts.
Based on these observations, we postulate that NRBCs
reach the heart via diapedesis through endocardial endothelium, whereas endothelial cells/angioblasts reach the
heart from the epicardial surface (Muñoz-Chápuli et al.,
2002; Poelmann et al., 2002), and that blood island-like
structures are formed by angioblasts establishing contact
with NRBCs, and angioblasts encircle NRBCs and assemble, forming primitive vascular vesicles or blood islands.
Some individual cells of erythroblastic lineage (which
exhibit Ter/119 antigen) (Ikuta et al., 1990) were found to
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RATAJSKA ET AL.
Fig. 4. A 12.5 dpc heart, semithin sections. Solitary nucleated red
blood cells within the subepicardium. a: NRBC is making contact with
angioblast (arrow). b: NRBC surrounded by myocardial cells and the
epicardial cells (arrow). c: NRBC outside a vascular vesicle (arrow).
Magnifications: 380⫻.
cell precursors when transplanted to fetal liver (PérezPomares et al., 2004). Since the presence of hematopoietic
cells in the proepicardium had been previously suggested
by Virágh et al. (1993), we wanted to verify this notion.
Detailed ultrastructural analysis of the proepicardium allowed us to disprove the hypothesis that NRBCs are supplied by the proepicardium, the presumed source of angioblasts/endothelial cells. Neither red blood cells (bearing
Ter/119 antigen) nor any cell of hematoblastic activity
(bearing CD34 or CD45 antigen) (Wood et al., 1997) has
been detected in the proepicardium. Thus, we come to the
conclusion that the authors (Virágh et al., 1993) had considered the proepicardium as an organ that maintained its
properties while attached to the heart and spread on its
surface. According to current definition, the proepicardial
organ is understood as a transient tissue that protrudes to
the pericardial cavity (Männer, 1999; Männer et al., 2001;
Pérez-Pomares et al., 2002; Kattan et al., 2004). When the
tissue attaches to the heart, it is no longer considered to be
the proepicardium but the epicardium and its derivative,
subendocardial mesenchyme. Thus, the presence of hematoblasts or NRBCs detected by Virágh et al. referred to
their location in the subepicardium at the onset of heart
vascularization (Virágh and Challice, 1981; Virágh et al.,
1993), which has been consistent with our observation.
A possibility that NRBCs enter the myocardium from
the epicardial surface had been suggested before (Virágh
et al., 1990). Blood cells have been assumed to be enveloped by endothelial cell progenitors and internalized to
the subepicardium and subsequently to myocardium. We,
however, were unable to detect any blood cells floating
freely within the pericardial cavity when the pericardium
was intact during isolation of the embryo (Fig. 2a). The
only cells detected within the pericardial cavity formed
vesicles deriving from the proepicardium and from the
pericardial mesothelium.
Thus, we postulate that NRBCs enter the heart independently from angioblasts. Subsequently, both kinds of
cells move toward each other and assemble to form blood
island-like structures, which are initially located in the
subepicardium. Since the distance from the endocardium
to the epicardium at the onset of heart vascularization is
short (due to the very thin myocardial wall consisting of
two to three layers of cardiocytes with deep trabecular
invaginations) (Manasek, 1968), the movement of erythroblasts to the heart surface does not take a long time.
Another possibility of RBC derivation in embryonic
heart at the onset of vascularization might be in situ
differentiation from progenitor cells that had migrated
into the heart before. The embryonic heart has been postulated to be the hematopoietic organ in previous papers
by Virágh et al. (1990) and Kálmán et al. (1995). There are
several reports indicating that hematopoiesis within the
embryo is strictly related to the areas of vasculogenesis
(Pardanaud et al., 1989). We were unable, however, to
demonstrate the presence of hematopoietic stem cells in
the embryonic heart neither by the use of TEM or by
immunohistochemical staining with antibodies to HSC
antigens. We also could not find any blood island exhibiting a pattern of cellular assembly similar to a yolk sac
blood island (Haar and Ackerman, 1971). Our study indicates that the embryonic heart supplies only new erythroblasts owing to their proliferative capacity within the
primitive vascular vesicles at the time before coronary
vessels are connected to the aorta (Fig. 3). It is doubtful
BLOOD ISLAND-LIKE STRUCTURES
Fig. 5. An 11.5 dpc heart. Nucleated red blood cells located subendocardially and not accompanied by angioblasts. The lumen of the
ventricle marked with asterisks. a: Semithin section stained with toluidine blue. Red blood cells marked with an arrow. b–d: Frozen sections
double-stained with anti-Ter/119 (brown) and anti-PECAM1 (blue) antibodies. Erythroblasts located subendocardially (arrows) not accompanied by PECAM1⫹ angioblasts. However, they adjoin the endocardial
Fig. 6. An 11.5 dpc heart. NRBC while passing through the endocardial endothelium (en). The lumen of the ventricle marked with an
asterisk. Magnification: 4,500⫻.
229
endothelial cells. The latter are PECAM1⫹. e: Ultrastructure of subendocardially positioned nucleated red blood cells. Erythroblasts adhere to
endocardial endothelium and are surrounded by myocardial cells (c).
Magnifications: 380⫻ (a); 350⫻ (b– d); 4,500⫻ (e).[Color figure can be
viewed in the online issue, which is available at www.interscience.
wiley.com].
Fig. 7. A 13 dpc heart. NRBC within the subepicardial space, partially encircled by the epicardial epithelium (ep). Magnification: 6,000⫻.
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RATAJSKA ET AL.
Fig. 8. An 11.5 dpc heart. a: Cellular cluster of angioblasts and a
nucleated red blood cell at the process of coming in contact. Erythroblast exhibits pseudopods, which is indicative of the cell’s movement. b:
Nucleated red blood cells adjoining angioblasts. Angioblasts do not form
a vessel lumen yet (absence of the intercellular junctions). However, they
encircle red blood cells. Magnifications: 6,600⫻ (a); 5,800⫻ (b).
Fig. 9. An 11 dpc heart. Subendocardial space with NRBC. NRBC is
being encircled by an angioblast process. Magnification: 4,800⫻.
that the embryonic heart possesses a hematopoietic activity since this activity is always associated with production
of many descendent cells, as has been demonstrated in the
fetal yolk sac (Haar and Ackerman, 1971; Tavassoli,
1991), aorta-gonads-mesonephros (AGM) region of the embryo, and fetal liver (Dzierzak et al., 1998; Dzierzak, 2002,
2003).
Fig. 10. An 11 dpc heart. a: TEM of an erythroblast in a blood
island-like structure with a halo around the nucleus (white arrow). b:
Erythroblast from the blood stream of the same stage of development
with nucleus and organelles in the cytoplasm. Magnifications: 5,000⫻
(a); 4,800⫻ (b).
Since formation of blood island-like structures occurs
throughout the prenatal life (Rongish et al., 1994), it is
possible that red blood cells (nucleated or enucleated)
enter the embryonic heart also at later stages of development. We were unable, however, to study the mode of their
entrance after the formation of the connection between the
coronary system and the peripheral circulation, i.e., after
13.5 dpc. A possible mode of red blood cell entrance to the
heart at later stages of coronary system development
might be simply via diapedesis from capillary plexus.
Our study suggests that NRBCs enter the heart from
the endocardium, whereas studies by other authors (Poelmann et al., 1993) have decisively indicated that the endocardial endothelial cells do not take part in the formation of coronary vasculature. Although the endocardial
endothelium does not have a potential to differentiate into
endothelial cells of coronary vasculature, it may exhibit
properties of route via which NRBCs migrate to the myocardial wall.
Thus, it is highly possible that NRBCs arriving from the
endocardium assemble together with angioblasts coming
from the epicardium. Studies by Tomanek et al. (1996,
1999) have demonstrated that there is an epicardial-endocardial gradient of density of newly formed vessels, as
well as angiogenic growth factors such as VEGF. Precursors of coronary vasculature arrive to the heart from the
BLOOD ISLAND-LIKE STRUCTURES
epicardial surface (Muñoz-Chápuli et al., 1996, 2002; Gittenberger-de Groot et al., 1998; Pérez-Pomares et al.,
2002; Poelmann et al., 2002) and settle initially in the
subepicardium, which is consistent with the highest density of new vessels in this area. These statistic measurements are not affected by single scattered NRBCs without
angioblasts found in the subendocardium.
Thus, we postulate the subsequent sequence of events:
erythroblasts are generated within the hematopoietic foci
of the 9 –13 dpc embryo: yolk sac, AGM (Muller et al.,
1994; Medvinsky and Dzierzak, 1996; Jaffredo et al., 1998;
de Bruijn et al., 2002), placenta (Alvarez-Silva et al.,
2003), and liver (Dzierzak et al., 1998), and circulate
freely within the peripheral circulation. Subsequently,
some of them adhere to endocardial surface and enter
myocardium via diapedesis and finally they move toward
the nearest angioblasts. Angioblasts arrive from the epicardium, as has been postulated previously by several
authors (Poelmann et al., 1993; Pérez-Pomares, 2002).
The entrance of NRBCs to myocardium takes place concomitantly with angioblasts, i.e., after 10.5 dpc (after the
epicardium had covered the majority of the heart’s surface).
It is not known whether the presence of nucleated/enucleated red blood cells within the embryonic heart before
the coronary system is connected with the aorta is of any
significance. NRBCs found in blood island-like structures
and within the vascular vesicles of the embryonic heart
are certainly a source of erythrocytes within the peripheral circulation from the time point when blood starts to
circulate within the coronary system. They may also be a
source of certain paracrine substances to the adjacent cells
(angioblasts) in vasculogenic events.
ACKNOWLEDGMENTS
The technical assistance of Maria Michniewska and
Anna Podbielska is greatly appreciated.
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