Download Circulation is established in a stepwise pattern in the mammalian

Plenary paper
Circulation is established in a stepwise pattern in the mammalian embryo
Kathleen E. McGrath, Anne D. Koniski, Jeffrey Malik, and James Palis
To better understand the relationship between the embryonic hematopoietic and
vascular systems, we investigated the
establishment of circulation in mouse embryos by examining the redistribution of
yolk sac–derived primitive erythroblasts
and definitive hematopoietic progenitors.
Our studies revealed that small numbers
of erythroblasts first enter the embryo
proper at 4 to 8 somite pairs (sp) (embryonic day 8.25 [E8.25]), concomitant with
the proposed onset of cardiac function.
Hours later (E8.5), most red cells remained in the yolk sac. Although the
number of red cells expanded rapidly in
the embryo proper, a steady state of ap-
proximately 40% red cells was not reached
until 26 to 30 sp (E10). Additionally, erythroblasts were unevenly distributed within
the embryo’s vasculature before 35 sp.
These data suggest that fully functional
circulation is established after E10. This
timing correlated with vascular remodeling, suggesting that vessel arborization,
smooth muscle recruitment, or both are
required. We also examined the distribution of committed hematopoietic progenitors during early embryogenesis. Before
E8.0, all progenitors were found in the
yolk sac. When normalized to circulating
erythroblasts, there was a significant enrichment (20- to 5-fold) of progenitors in
the yolk sac compared with the embryo
proper from E9.5 to E10.5. These results
indicated that the yolk sac vascular network remains a site of progenitor production and preferential adhesion even as
the fetal liver becomes a hematopoietic
organ. We conclude that a functional vascular system develops gradually and that
specialized vascular–hematopoietic environments exist after circulation becomes
fully established. (Blood. 2003;101:
1669-1676)
© 2003 by The American Society of Hematology
Introduction
A functional circulatory system is an early requirement for survival
and growth of the mammalian embryo and is the first organ system
to develop in the embryo.1 The circulatory system is composed of
vascular, hematopoietic, and cardiac components, each formed
from discrete regions of mesoderm. The first endothelial cells and
blood cells are generated in yolk sac blood islands beginning at
embryonic day 7 (E7.0) in the mouse. By E8.0, thousands of
nucleated primitive red blood cells have formed within a vascular
plexus in the yolk sac.2-4 Concurrently, the aorta and the peristaltic
beating heart tube form in the embryo proper. In the next 36 hours,
there is a remarkable increase in complexity of vascular and
hematopoietic systems. The vascular plexus remodels and expands
into an arborized network of specialized arteries and veins.
Primitive erythroblasts from the yolk sac continue to divide and
mature, and a second wave of hematopoiesis creating definitive
(adultlike) progenitors originates in the yolk sac.5,6 Thus, the early
vascular system is the hematopoietic environment for primitive and
definitive lineages until the specialized stromal microenvironment
of the fetal liver (beginning E10), and later the adult bone marrow,
is available. However, the nature of any specific interactions
between early embryonic hematopoietic cells and endothelial cells
is unclear.
A peristaltic beating heart tube and the connection of the yolk
sac and embryo proper vasculature have generally been considered
sufficient evidence for functional circulation. This is thought to
occur at E8.5 (8-10 somite pairs [sp]).7,8 More recently, unpub-
lished results have been used to contend that hematopoietic
progenitors “freely circulate between extra- and intra-embryonic
compartments” as early as E8.25 or 5 sp.9 In contrast, we found that
definitive hematopoietic progenitors remain almost entirely restricted to the yolk sac as late as E9.25.5 This suggests that the
circulation is initially very limited or that the yolk sac is a
specialized hematopoietic environment despite circulation. Studies
reported here present evidence supporting both these possibilities
by taking advantage of the initial asymmetry of mammalian blood
synthesis to more carefully determine the onset of circulation in
staged mouse embryos.
From the Center for Human Genetics and Molecular Pediatric Diseases,
Department of Pediatrics, University of Rochester, NY.
Reprints: Kathleen E. McGrath, Center for Human Genetics and Molecular
Pediatric Disease, University of Rochester, Box 703, 601 Elmwood Ave,
Rochester, NY 14642; e-mail: [email protected].
Submitted August 16, 2002; accepted October 1, 2002. Prepublished online as
Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-08-2531.
Supported by National Institutes of Health grant R01 HL59484 and American
Heart Association grant 0130503T.
BLOOD, 1 MARCH 2003 䡠 VOLUME 101, NUMBER 5
Materials and methods
Embryo dissection and cell dissociation
ICR mice (Taconic, Germantown, NY) were mated overnight, and vaginal
plugs were checked the following morning (E0.5). Maternal tissues and
Reichert membrane were removed, and the yolk sac was rapidly detached
from the embryo proper. For numerical analysis of erythroblasts in E9.0 and
later embryos, dissections were performed on concepti in individual dishes
while embryonic circulation was intact. Although there was no discernible
flow from dissected yolk sacs, blood did continue to flow from the umbilical
arteries of E9.5 and older embryos. Accordingly, all blood was collected
from the dish and was included in the embryo proper values. Occasional
contaminating maternal red blood cells were easily distinguished by size
from the primitive erythroblasts and were excluded from cell counts.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2003 by The American Society of Hematology
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MCGRATH et al
Tissues were dissociated at room temperature in a 1:30 dilution of 0.25%
trypsin (Worthington Biochemical, Lakewood, NJ) in phosphate-buffered
saline (PBS)/1 mM EDTA (ethylenediaminetetraacetic acid) with occasional trituration by pipette to aid in digestion. Once samples were
dissociated into single cells, an equal volume of Iscove modified Dulbecco
medium (IMDM; Gibco BRL, Grand Island, NY)/20% plasma-derived
serum (PDS; Animal Technologies, Tyler, TX) was added, and cells were
centrifuged and resuspended in IMDM/20% PDS. Total cell numbers and
viability were determined by trypan blue staining (Sigma, St Louis, MO).
Hematopoietic progenitor assays
Dissociated cells were plated in methylcellulose, as previously described,5
but IMDM and 0.8% methylcellulose were used with the inclusion of the
following cytokines: interleukin-3 (IL-3; 20 ng/mL), IL-6 (20 ng/mL), stem
cell factor (SCF; 60 ng/mL), granulocyte macrophage (GM; 2.5 ng/mL) (all
from R&D Systems, Minneapolis, MN), and erythropoietin (EPO; 2 U/mL;
Amgen, Thousand Oaks, CA). Cultures were incubated at 37°C, 5% CO2.
Erythroid–colony-forming units (CFU-Es) were counted after 3 days of
culture, and erythroid blast-forming unit (BFU-E) and macrophage–colonyforming unit (CFU-mac) colonies were counted after 7 days of culture.
Culturing of high proliferative potential–colony-forming cells (HPP-CFCs)
was carried out on agar plates, as previously described.10
In situ hybridization
Embryos were fixed in 4% paraformaldehyde in PBS overnight at 4°C,
rinsed with PBS, dehydrated in methanol, bleached with 5% hydrogen
peroxide in methanol for 5 hours, and then rinsed and stored in methanol.
Digoxigenin-labeled antisense RNA probes corresponding to nucleotides
74 to 421 of ␤H1-globin mRNA (GenBank accession no. J00417) were
synthesized using the DIG RNA Labeling Kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions. Whole-mount in situ
hybridization was carried out as described by Wilkinson11 with the
following modifications: no proteinase K or RNase A treatments were
performed, embryos were permeabilized with 3 washes, 10 minutes each, of
RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8, 1 mM EDTA, 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS])12;
hybridization buffer was at pH 4.5 with 0.1% Tween instead of SDS, and
probe was added to a final concentration of 0.5 ␮g/mL. Additionally,
posthybridization washes were simplified to 3 washes, 30 minutes each, at
70°C each of solution 1 and solution 3, followed by 3 washes, 5 minutes
each, with TBST (0.14 M NaCl, 2.7 mM KCl, 25 mM Tris-HCl, pH 7.5,
0.1% Tween) at room temperature. After nitroblue tetrazolium/x-phosphate
5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) staining, embryos were
dehydrated in methanol and rehydrated in TBST to darken the stain. Stained
embryos were cleared in 50% glycerol in TBST, followed by 80% glycerol
and 0.02% sodium azide in TBST.
Immunohistochemistry
Embryos were fixed and bleached as for in situ hybridization. After
rehydration into PBT (PBS, 0.1% Tween 20), embryos were blocked for 2
hours in PBSMT (PBS, 3% instant milk, 0.1% Triton X-100) then incubated
overnight at 4°C with 1:200 dilution of rat antimouse platelet endothelial
cell adhesion molecule-1 (PECAM-1; BD Biosciences PharMingen, San
Diego, CA) in PBSMT. Embryos were washed 3 times for 5 minutes and
then 5 times for 1 hour in PBSMT at 4°C. Embryos were then incubated
with secondary antibody and washed as for primary antibody. The
secondary antibody was either 1:100 dilution of horseradish peroxidase–
conjugated goat antirat immunoglobulin G (IgG) or 1:200 dilution of
alkaline phosphatase-conjugated goat antirat IgG (both from Jackson
ImmunoResearch, West Grove, PA). For horseradish peroxidase detection,
embryos were incubated for 20 minutes in 0.3 mg/mL p-dimethylaminoazobenzene (DAB), 0.05% NiCl2 (Vector Labs, Burlingame, CA) in PBT, then
developed with the addition of H2O2 to a concentration of 0.03%. The
staining reaction was stopped by a PBT rinse. For alkaline phosphatase
staining, the embryos were developed and cleared as for in situ hybridization.
Benzidine and o-dianisidine staining of
hemoglobin-containing cells
By 5 sp, erythroblast hemoglobin content was sufficient to allow detection
by benzidine staining of dissociated cells, as previously described.13
Hemoglobin-containing cells in intact embryos were visualized using
o-dianisidine (Sigma) staining performed as described by O’Brien,14 with
the following modifications: dissections were performed in PB-1 with 50
mM KCl added to stop the heartbeat.15 Embryos were then rinsed in PB-1,
stained in o-dianisidine solution with hydrogen peroxide reduced to 0.15%,
postfixed in 4% paraformaldehyde in PBS for 20 minutes, rinsed in PBS,
and cleared in glycerol as for in situ hybridization. This method directly
detects hemoglobin-containing cells and avoids difficulties associated with
the asynchronous decrease in ␤H1-globin message that occurs in red blood
cells beginning at E9 (data not shown).
Results
Red blood cell movement begins in 4 to 6 sp embryos but is
extremely limited
Although it is generally assumed that 5 sp (E8.25) embryos
demonstrate functional circulation, the onset and extent of blood
flow have not been specifically examined. One of the difficulties in
determining the timing of developmental events in the early mouse
embryo is the variability of developmental stage within a particular
embryonic day.16,17 This is caused by 2 factors. First, variability in
developmental stages between and even within litters can be
extensive. Second, embryos develop rapidly, and profound changes
occur within a few hours. Accordingly, we used the number of
somite pairs to more precisely and accurately stage individual
embryos. Somite pairs are formed in the murine embryo between
E8 and E10 at an approximate rate of 1 every 1.8 hours.17 To
determine the kinetics of circulation, we took advantage of the fact
that definitive hematopoietic progenitors do not produce circulating
red blood cells until E12, so early embryo red blood cells are
derived from primitive erythroid progenitors confined to the yolk
sac.5,6 Therefore, circulation could be detected as the redistribution
of primitive nucleated red blood cells from yolk sac into the
embryo proper. Yolk sac–derived primitive erythroblasts are also
distinguished from the fetal liver–derived definitive erythrocytes
by expression of embryonic hemoglobin.6 We used in situ hybridization to embryonic-specific ␤H1 globin mRNA to detect erythroblasts before they accumulated enough hemoglobin to be detected
by direct staining by benzidine. Vascular development during
embryogenesis was assessed by PECAM immunohistochemistry.18
We chose not to use the earlier marker of endothelial potential,
vascular endothelial growth factor receptor-2 (VEGFR-2) (flk-1),
because it is also expressed in early progenitors of blood and
smooth muscle lineages and is down-regulated in many mature
vascular beds.4,19
From 0 to 3 sp, erythroblasts are not only restricted to the yolk
sac but are present in a narrow band of blood islands forming at the
most proximal region (farthest from the embryo proper) of the yolk
sac walls (arrowhead, Figure 1A,C). We sought to determine
whether vascular development in the yolk sac was similarly limited
to blood islands. At presomite stages, the first PECAM-expressing
cells are in the blood islands (data not shown).4,18 As somatogenesis
begins, vascular development remains asymmetric in the yolk sac
and is centered at the blood islands (Figure 1B). Simultaneously,
angioblasts within the embryo proper begin to differentiate and
coalesce into the aortae (Figure 1B, arrow). By 3 sp, vascular
development has spread throughout the yolk sac, and extended
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STEPWISE ONSET OF CIRCULATION IN MAMMALIAN EMBRYO
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Figure 1. Asymmetric blood formation and first blood flow in early somite pair embryos. In rodents, the embryo proper develops within the amnionic sac, which itself is
within the yolk sac. In this figure, the yolk sac was photographed with the embryo proper inside, except for panels D and I, where the embryo proper was dissected free of
amnion and yolk sac. (Top row) Blood flow was monitored by the redistribution of yolk sac–derived primitive erythroblasts detected by in situ hybridization to the
embryonic-specific ␤H1 globin mRNA. (Bottom row) Endothelial development was delineated by PECAM immunohistochemistry. No staining was seen in stage-matched
negative controls of in situ hybridization or immunohistochemistry (see panels I and J for examples). (A-B) At 1 sp, red blood cells were restricted to blood islands (red
arrowheads) in the proximal region (farthest from the embryo proper), where vascular development was most prominent. The yolk sac vascular plexus centered at the blood
islands and the embryo proper aortae (arrow) had begun to form (h indicates head; t, tail). (C-D) By 3 sp, blood was still restricted to the proximal yolk sac, whereas vascular
development had spread throughout the yolk sac, and extended aortic tubes were visible (arrow). (E-G) At 4 sp, the first few dispersed red blood cells were observed (red
arrow) in the distal yolk sac (E) and in the embryo proper (G). At this stage, a well-developed vascular plexus extended throughout the distal yolk sac (F), forming the vitelline
connections (v) with the vasculature of the embryo proper (H). Stars in panel H indicate sections of the forming cardinal vein. Somite pairs in panels I and J are marked with
small squares. Original magnification A-J, ⫻ 75.
aortic tubes are visible (Figure 1D); however, red blood cells
remain restricted to the blood islands of the proximal yolk sac
(Figure 1C). These findings indicate that there are hemangiogenic
and angiogenic regions within the yolk sac.
Beginning at 4 sp and later, we observed some embryos with
erythroblasts in the distal yolk sac, though they were not in large
clusters as seen in proximal blood islands. Instead, dispersed single
and small groups of erythroblasts were present within the vascular
plexus throughout the distal yolk sac (Figure 1E-F). These observations suggest that primitive erythroblasts form in blood islands and
then move into more distal regions of the yolk sac. Coincident with
the first appearance of red blood cells in the distal yolk sac, we
found a few red blood cells in the embryo proper of some 4 sp
embryos (Figure 1G). These cells were generally seen in the head
and tail regions, where embryonic and yolk sac vasculatures meet
anterior to the atrium and at the posterior extreme of the aortae
Figure 2. Limited blood flow in E8.25 to E8.75 embryos. In the top row, embryos proper and yolk sacs remain attached. In the bottom row, embryos proper were dissected
free of extraembryonic tissues. Before 8 sp, an embryo proper is initially curved with its back arched. From 8 to 12 sp, the embryo proper then turns into the more traditional fetal
position. Distribution of ␤H1 globin mRNA-containing erythroblasts was detected by in situ hybridization (A-F), and distribution of PECAM-expressing endothelial cells was
detected by immunohistochemistry (G-H). (A-B) At 6 sp, most of the red blood cells were in the yolk sac and particularly concentrated at the proximal edge (red arrowhead). The
few erythroblasts (red arrows) in the embryo proper were seen in the head and aorta. (C-D) At 8 sp, more red blood cells were seen throughout the yolk sac and within the aorta
and head regions. (E-H) By 12 sp, while most blood was still in the yolk sac, increased numbers of red blood cells were seen in the embryo proper and were found throughout
the length of the aortae. However, blood was not seen throughout the entire embryonic endothelial network (compare panels F and H). For example, blood was rarely seen in
the intersomitic arteries (s) that were observed at this time. v indicates vitelline connections of yolk sac and embryo proper vasculature. Original magnification A-D, ⫻ 75;
E-H, ⫻ 50.
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Figure 3. Highly regulated timing of initial stages of blood flow in murine
embryos. Individual embryos were divided into 5 groups based on their yolk sac (YS)
and embryo proper (EP) red blood cell patterns (indicated in red). The somite pair
range of embryos that had each pattern is indicated.
(Figure 1F,H). Occasionally, a few erythroblasts were also observed in the central region of the aortae. Although this represented
the beginning of blood flow into the embryo proper, it was an
extremely small fraction of the total red blood cells (fewer than 20
of 10 000 red blood cells). Indeed, circulation throughout the
embryo proper is not possible because the cardinal vein has not yet
formed (Figure 1H, stars).
Blood flow increases only slightly from E8.5 to E8.75
Beginning at 8 sp (E8.5), we observed an increase in numbers of
erythroblasts in the head vessels and anterior aortae (compare
Figure 2B,D). Concurrently, red blood cells were more extensively
localized in the distal yolk sac (compare Figure 2A,C). Despite the
increase in numbers of red blood cells in the embryo proper from 8
to 12 sp, most red blood cells remained in the yolk sac (Figure
2E-F). Additionally, the distribution of red blood cells within the
embryo was limited to the sites that most directly receive blood
flow from the heart—head vessels and aortae. Interestingly,
PECAM staining demonstrated that vascular development was
more widespread than the distribution of red blood cells during this
stage. In particular, PECAM staining of intersomitic arteries was
present hours before red blood cells were detected (Figure 2H).
These data indicate that though red blood cells do move into the
embryo proper at early somite stages (4 to 6 sp), for the next 12
hours the early cardiovascular system moves only a small fraction
of the total red blood cells to a subset of the developing endothelial
network of the embryo.
BLOOD, 1 MARCH 2003 䡠 VOLUME 101, NUMBER 5
small numbers of red blood cells were found in the distal yolk sac
and the embryo proper. Because primitive erythroblasts originate in
the yolk sac, their presence in the embryo proper indicates that
some type of blood flow has begun. Additionally, examination of
25 4 to 6 sp embryos revealed a strict correlation between the
presence of erythroblasts in the distal yolk sac and the presence of
erythroblasts in the embryo proper. This suggests that the appearance of red blood cells in the distal yolk sac is caused by the same
factors that cause the first movement of red blood cells into the
embryo proper. All embryos with 8 or more somite pairs had a
quantitative increase in numbers of red blood cells in the embryo
proper; by 15 sp, red blood cells could be found in the intersomitic
arteries closest to the heart. Changes in red blood cell distribution
in the embryo proper also correlated with a marked increase in
numbers of red blood cells observed in the distal yolk sac. The
precision of these transitions suggests an incremental acquisition of
circulatory function. Although the entire cardiovascular system
continues to mature during E8, the changes in red blood cell
distribution correlate well with dramatic changes in cardiac function that occur at 4 to 8 sp. Fusion of the heart primordia,
development of myofibrillar bundles, and initiation of contractions
occurs at 4 sp. The transition to a looped peristaltic beating heart
occurs by 8 sp.7,20,21
Increasing circulation within the embryo proper does not
achieve a steady-state density of red blood cells until E10
Our in situ analysis indicated that most primitive erythroblasts
were retained in the yolk sac even 12 hours after the first movement
of red blood cells into the embryo proper. We wanted to determine
when a significant proportion of red blood cells moved into the
embryo proper. Therefore, total cells and hemoglobin-containing
cells were counted from individual concepti separated into embryo
proper and yolk sac fractions. Over a 48-hour period, erythroblasts
continued to divide and mature within the vascular system, causing
an exponential increase in red blood cell numbers in the embryo
proper and the yolk sac (Figure 4A). There was also a redistribution
of red blood cells caused by circulation. At E8.5 (5-9 sp), there
were 100-fold more red blood cells in the yolk sac than in the
embryo proper. Twenty-four hours later, at E9.5 (20-24 sp), half the
red blood cells were in the embryo proper, and by E10.5 (35-40 sp),
10-fold more red blood cells were found in the embryo proper than
in the yolk sac (Figure 4A).
We reasoned that a functional end point for the establishment of
robust circulation is the provision of a sufficient density of red
blood cells to adequately oxygenate tissue. Therefore, we determined the density of red blood cells as the percentage of total cells
Changes in distribution of red blood cells are precisely
temporally regulated
To determine how tightly the timing of the onset of blood flow is
regulated in E8 embryos, we compared the distribution of red blood
cells in 57 embryos. Embryos were categorized by their spatial
patterns of erythroblasts (Figure 3, schematics). In the yolk sac, 3
patterns were seen: red blood cells confined to blood islands,
occasional dispersed distal cells, and diffuse distribution. Embryos
proper were assigned 1 of 5 patterns: no red blood cells, small
numbers of red blood cells, hundreds of red blood cells, thousands
of red blood cells, and red blood cells within intersomitic arteries.
As shown in Figure 3, there was a remarkable correlation of each
pattern within a narrow range of somite pair numbers, indicating
that the onset of circulation is precisely timed. Beginning with 4 sp
embryos and including all embryos at 6 sp (or within 3 hours),
Figure 4. Changes in red blood cell distribution and density in the embryo
proper and yolk sac. Embryos were dissected into embryo proper and yolk sac and
dissociated into single cells, and total cells and erythroblasts (benzidine positive)
were enumerated. (A) Absolute numbers of erythroblasts per tissue were plotted, and
(B) percentages of total cells that were erythroblasts were plotted. Error bars indicate
SEM (n ⫽ 5-13).
BLOOD, 1 MARCH 2003 䡠 VOLUME 101, NUMBER 5
that were erythroblasts in the yolk sac and embryo proper between
E8.0 and E10.5. The density of red blood cells in the yolk sac
increased and remained high through E10.5 (Figure 4B), consistent
with its highly vascular nature and previous reports of blood cell
density in the yolk sac.22 From E8.0 to E9.0, the density of red
blood cells in the embryo proper increased only slightly. However,
after E9.0, there was a sharp increase in density of erythroblasts
from 3% to 40%, reaching equilibrium by E10.0.
Distribution of red blood cells throughout the vascular system
of the embryo proper is also delayed until E10.5
In addition to sufficient red blood cell density, functional
circulation requires that blood flow in a circular loop throughout
the entire vascular system. Throughout E8, erythroblasts were
localized only in head vessels and the aortae (Figure 2). We
wanted to determine whether the sharp increase in embryo
proper red blood cell density, beginning at E9.0, was correlated
with erythroblasts now distributed throughout the entire endothelial network. Alternatively, the rapid increase in total red blood
cell density from E9 to E10 could be accompanied by the filling
of progressively more vascular beds with red blood cells. The
latter possibility has intriguing implications for inequities in
flow, shear stress, and oxygenation within the vascular system at
the time of extensive angiogenesis. We compared the distribution of red blood cells with the vascular pattern in stage-matched
embryos to distinguish between these possibilities.
By 24 sp (E9.5), the basic structure of the endothelial system
has changed from vascular plexuses connected to large aortae and
cardinal veins into an arborized network (Figure 5A). The arborized network continues to be elaborated, increasing in complexity through E10.5 (Figure 5C,E). From 25 to 35 sp, the red blood
cell pattern also increases in complexity (Figure 5B,D,F). Comparisons of developmentally matched embryos indicated that at 24 sp,
the red blood cells were still concentrated in a subset of the
available vascular system. However, by 35 sp, the complexities of
vascular and blood patterns were comparable. This change was
particularly evident along the dorsal regions of the embryo proper.
At 12 sp (E8.75), the initiation of angiogenesis was evident as
intersomitic arteries branched off the aortae between each somite
pair (Figure 2H, arrows). By E9.5 (24 sp), the venous component of
Figure 5. Distribution of red blood cells throughout the
embryo proper vasculature occurs by 35 sp (E10.5). The
pattern of the embryo proper endothelial and red blood cell
distribution was visualized in stage-matched embryos by
PECAM immunohistochemistry (A, C, E, G-I) and o-dianisidine staining of hemoglobin (B, D, F, J-L), respectively. To
facilitate comparison, the 12th sp is indicated by a dot, the
20th sp by the letter x, and the 25th sp by a star (h indicates
heart ventricle; lb, limb bud). (A, C, E) The extensive arborized vascular network evident in the head and ventricle
and between the somite pairs at E9.5 (24 sp) became
progressively more complex by E9.75 (28 sp) and E10.5 (35
sp). (B, D, F) Red blood cells initially more concentrated in a
subset of vessels at 24 sp became more evenly dispersed by
35 sp. Additionally, high levels of hemoglobin-containing cells
were seen in the fetal liver (arrow) as it became the primary
site of hematopoiesis by E10.5. (G-L) Along the back, paired
intersomitic arteries and veins meet at capillary networks of
increasing complexity between somite pairs during this time
period. Initially, red blood cells were observed in paired
intersomitic vessels in the anterior back, at mid positions in
deeper arteries only, and not at all between posterior somite
pairs. By 35 sp, red blood cells had penetrated intersomitic
vasculature throughout the back. Red and blue arrowheads
indicate intersomitic arteries and veins, respectively. Original
magnifications A-F, ⫻ 20; G-L, ⫻ 50.
STEPWISE ONSET OF CIRCULATION IN MAMMALIAN EMBRYO
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intersomitic vessels became evident as more superficial intersomitic veins branched off the cardinal vein and connected to the
vertebral arteries at the dorsal edge of the neural tube23 (Figure 5G,
red and blue arrowheads). As each somite pair was formed, the new
vascular loop was extended such that intersomitic vessels were
present between even the most posterior somite pairs. In contrast,
red blood cells were not found between the more posterior somite
pairs (for example, blood was seen only through 16 sp in a 24 sp
embryo; Figure 5J). In more anterior positions, stripes of red blood
cells were observed in deep intersomitic arteries and in superficial
intersomitic veins (Figure 5J, K, red and blue arrowheads, respectively). However, in the more posterior positions, a single stripe of
red blood cells was observed in the deeper intersomitic arteries. By
E10.5 (35 sp), erythroblasts were found between all somite pairs,
including in the superficial intersomitic veins (Figure 5L). Therefore, blood was not fully dispersed in the endothelial network of the
embryo proper until after E10.0. Additionally, there was a restriction of red blood cells to intersomitic vessels closer to the heart at
E9.5. This restriction was no longer observed by E10.5. This
closely followed the achievement of a plateau in the density of the
red blood cells within the embryo proper (Figure 4B). These data
suggest that from E9 to E10, there was a maturation of the
cardiovascular system required for blood to flow in a complete
circuit of the available vascular beds.
Hematopoietic progenitors do not circulate freely, even after
the onset of circulation
Along with primitive erythroblasts, the embryonic circulation also
contains the first progenitor cells for definitive hematopoiesis
arising in the yolk sac.5 Differentiation of definitive hematopoietic
progenitors during fetal and adult periods is dependent on interactions within the stromal microenvironments of the liver and the
bone marrow, respectively. In contrast, embryonic hematopoiesis is
thought to consist of free-floating cells in the vascular system.2 If
yolk sac–derived definitive hematopoietic progenitor cells were
free floating, they should have entered the circulatory system of the
embryo proper at the same rate as primitive erythroblasts. We
tested this hypothesis by measuring the numbers of hematopoietic
progenitors in the embryo proper when a significant proportion of
erythroblasts entered the embryo proper (starting at 20 sp; Figure
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MCGRATH et al
4A). Erythroblast numbers were used to quantify the extent of
circulation in individual embryos. We examined 3 unipotential
definitive hematopoietic progenitors (erythroid-producing BFU-E
and CFU-E and macrophage producing CFU-mac) and a multilineage HPP-CFC found exclusively in the yolk sac before 15 sp.5,10
Enrichment of progenitors in the yolk sac versus the embryo proper
was calculated by dividing the proportion of progenitors in the yolk
sac by the proportion of erythroblasts there. If progenitors moved
within the vascular system as freely as erythroblasts, this fold
enrichment of progenitors would be 1. Because our question
focused on progenitors within the yolk sac and embryo proper
vascular systems, the developing liver buds were removed from
embryos with 35 sp or more and were cultured separately. As
expected, at 35 to 40 sp, we observed a high level of enrichment of
BFU-Es and CFU-macs in the liver versus the embryo proper (70to 30-fold, respectively; data not shown). However, because of
their small size, the early liver buds still contained only a minority
of the total progenitors in the conceptus at these stages.
We found a significant enrichment of progenitors in the yolk sac
compared with the embryo proper at all stages examined (Figure
6). For BFU-Es and CFU-macs, enrichment was highest at 19 to 23
sp, but it persisted even as the liver became the predominant site of
hematopoiesis (35 sp). As previously reported, CFU-Es were not
observed before 25 sp5 but, once detected, were also enriched in the
yolk sac. Interestingly, the enrichment of CFU-Es in the yolk sac
was consistently lower than that of its own progenitors, BFU-Es.
This suggests that BFU-Es continue their preferential association
with the yolk sac as they mature into CFU-Es but that this specific
interaction decreases. However, decreasing yolk sac association
with maturation cannot be a universal characteristic because the
multilineage progenitor, HPP-CFC, actually circulates more extensively than the unilineage progenitors at 19 to 23 sp. Thus, though
all progenitors examined were enriched in the yolk sac, there are
indications of quantitative differences in the association with the
yolk sac. Enrichment in the yolk sac could be attributed to the
continued production of progenitors from fixed cells associated
with the vascular system or to the preferential adherence of
circulating progenitors to yolk sac endothelium. In either case,
these data are not consistent with all definitive hematopoietic
Figure 6. Hematopoietic progenitors are retained in the yolk sac despite
circulation. Numbers of BFU-E, CFU-E, CFU-mac, and HPP-CFC hematopoietic
progenitors and erythroblasts in the yolk sac versus the embryo proper were
determined. Fold enrichment was calculated as the ratio of progenitors in yolk sac to
embryo proper divided by the ratio of erythroblasts in the yolk sac to embryo proper. If
progenitors circulated as freely as erythroblasts, the expected enrichment would have been
1. However, significantly more of all 3 types of progenitors were found in the yolk sac than
would be expected, even as the fetal liver was becoming hematopoietic (E10.5). Three to 4
embryos were pooled for each experiment, and 3 to 6 independent experiments were
performed for each time point. Error bars indicate SEM.
progenitors existing as free-flowing cells within the yolk sac.
Rather, our findings suggest that hematopoietic progenitors specifically interact with a subset of the vascular bed, even after extensive
circulation and liver hematopoiesis have been established.
Discussion
Onset of circulation
Ultimately the determination of when embryonic circulation begins
is dependent on the definition of circulation. If circulation is
defined simply as red blood cell movement within vessels, then the
onset of circulation occurs in the mouse embryo within a narrow
window of time between 4 and 6 sp, during E8.25. However, this
definition fails to incorporate connotations of circulation critical for
function, including the movement of sufficient blood in a circuit to
oxygenate all tissues. Results reported here demonstrate a stepwise
acquisition of characteristics of circulation over 36 hours of
embryogenesis in the mouse. From E8.25 to E8.5 (4-7 sp),
circulation is characterized by the movement of a few yolk
sac–derived erythroblasts into the aortae of the embryo proper. The
first movements of erythroblasts correlate temporally with the
formation of a beating heart tube and linkage of embryonic and
yolk sac vascular systems.3,7,20,21,57 These data are consistent with
reports of the first erythroblasts entering the allantoic vasculature
(contiguous with the distal end of the aortae) at 6 to 10 sp.22 At E8.5
to E9.0 (8-15 sp), we found evidence of increased flow as red blood
cells penetrated further into the embryo proper arterial system.
Nonetheless, only one tenth of the yolk sac–generated red blood
cells entered the embryo proper by E9.0, though the embryo proper
had 10 times more total cells than the yolk sac. These data suggest
that the early cardiovascular system is insufficiently developed to
establish vigorous circulation throughout the E8 conceptus.
Between E9 and E10, we observed a rapid increase in the extent
of circulation so that by E10.5 (35 sp), erythroblasts were dispersed
throughout the entire vascular system, and a steady state density of
red blood cells within the embryo proper was achieved. The start of
increased blood dispersal seen at E9.25 temporally correlated with
a fundamental change in the structure of the vascular network.
Between 15 and 25 sp, the netlike vascular plexuses and large
aortae and cardinal veins were transformed by angiogenesis into an
arborized vascular system (Figure 2H, 4A).8,24 Two additional
major changes in the cardiovascular system, formation of a
chambered heart and specialization of vessel walls with recruitment of vascular smooth muscle cells, occur later in embryogenesis. The heart tube begins to remodel into chambers only at E11,
and recent data suggest that cardiac function increases steadily,
without major transitions, from E8.25 to E10.5.25,57 Likewise,
vascular smooth muscle cells only begin to differentiate along a
limited area of the aorta at 30 sp,26,27 and the expression of
transcripts for certain critical smooth muscle proteins does not
begin until E10.28 In the context of these observations, our data
suggest that a major factor in the achievement of robust circulation
at E10 is the maturation and remodeling of the vascular network.
Our results imply, but do not prove, that functional circulation is
not achieved until E10, concomitant with angiogenesis. This
hypothesis is supported by the fact that embryos mutant in genes
necessary for initial establishment of the endothelial networks
(VEGFR-2), red blood cell synthesis (GATA1, LMO2, and Tal1),
and regular heartbeat (NCX1) do not die before E9.5.29-33 Furthermore, these mutant embryos do not die earlier than embryos with
BLOOD, 1 MARCH 2003 䡠 VOLUME 101, NUMBER 5
STEPWISE ONSET OF CIRCULATION IN MAMMALIAN EMBRYO
defects blocking later angiogenesis or heart maturation (Tie2,
EphrinB2, and MEF2C).24,34,35 Further delineation of how vascular
remodeling, smooth muscle recruitment, and cardiac development
specifically affect circulation awaits mutations altering only one of
these processes.24,33-37
Vascular remodeling may require early circulation given that
an embryo that does not establish a normal heartbeat because of
a mutation of cardiac sodium–calcium exchange is defective in
angiogenesis.33 An interesting implication of our data is that
angiogenic remodeling that creates an arborized vascular structure between E9 and E10 is initiated under conditions of uneven
circulation. Shear stress and oxygenation are known to be potent
regulators of angiogenesis in the adult.37 The data presented
suggest that inequities in these factors exist during the period of
angiogenic remodeling and that the arterial vessels closest to the
heart experience blood flow before distal arterial vessels or the
venous system do. Whether this discrepancy is recognized and
used during development of the vascular system remains to be
explored.
Asymmetry of the yolk sac
The yolk sac is a deceptively simple bilayer structure of
mesoderm and visceral endoderm. Visceral endoderm provides
the embryo with liver and gut functions, including synthesis of
serum proteins.2 Recently, the visceral endoderm has been
shown to be a potent developmental regulator of specific
anterior embryonic structures,38 and it may also be critical in
directing yolk sac mesoderm fates.39,40 It has been generally held
that during the initial formation of the vascular system, the yolk
sac mesoderm is hemangiogenic, whereas the embryo proper
mesoderm is angiogenic.8,41,42 Our work identifies hemangiogenic and angiogenic regions within the yolk sac. Large pools of
red blood cells and sheets of endothelial cells—that is, blood
islands—develop by E8.0 in the proximal region of the yolk sac.
In contrast, vasculogenesis occurs without visible hematopoiesis within the distal yolk sac and embryo proper. Indeed, red
blood cells are not found in the distal yolk sac unless red blood
cell movement has been initiated. Drake and Fleming4 found a
similar asymmetry of distal versus proximal yolk sac using
immunohistochemical analysis of early vascular markers. The
yolk sac later displays additional asymmetries in vascular
remodeling, including arterial versus venous specification.43
Thus, the yolk sac has apparently compartmentalized processes
central to the development of hematopoietic and endothelial fates.
1675
Interconnections of endothelial and hematopoietic
development
Increasing evidence supports the intertwining of endothelial and
hematopoietic fates. Marker studies have found extensive overlap
in gene expression in the earliest progenitors of both lineages,
including LMO2, Tal1, VEGFR-2, Tie2, and CD34.4,44-47 Mutational analyses in mouse embryos have confirmed that the normal
development of hematopoietic and vascular systems require functional LMO2, Tal1, and VEGFR-2 genes.44,47-49 There are also
spatial and temporal correlations between the emergence of the first
blood cells and endothelial cells in blood islands of the yolk sac.
The most direct evidence of a common hemangioblast progenitor is
the isolation of bipotential cells during the development of
embryoid bodies from embryonic stem cells.50 The association of
vascular and hematopoietic fates continues in later development as
the first hematopoietic stem cells capable of engrafting an adult
recipient are associated with the aorta and omphalomesenteric
arteries at E10.5 in the mouse.51-53 This has led to the hypothesis
that the adult hematopoietic system arises from a hemogenic
endothelium with the embryo proper.42 In the adult, bone marrow is
a source of hematopoietic stem cells and recently identified
circulating endothelial stem cells.54,55 Although the precise relationship between endothelial and hematopoietic potential in the bone
marrow remains undefined, significant plasticity of cells fates can
be uncovered experimentally.41,42,56 The results presented here
support an additional relationship between hematopoietic and
endothelial cells that occurs normally in the embryo. Before bone
marrow or fetal liver stromas are available as hematopoietic
microenvironments, observations of blood island development
have led to the impression that embryonic blood cells form and
float freely within the vascular network. No specific microenvironmental role has been proposed for endothelial cells. In contrast, we
have observed that hematopoietic progenitors remain associated
with a specific vascular bed (ie, yolk sac) despite maturation of the
vascular system, onset of circulation, and colonization of the fetal
liver. The identification of specific temporal–spatial associations of
endothelial and hematopoietic cells in the embryo should facilitate
future studies regarding the molecular underpinnings of the interactions between these systems.
Acknowledgments
We thank Dr Joe Miano, Dr Laurie Milner, and Dr Colin Phoon for
critically reading the manuscript.
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Erratum
In the article by Ruzickova et al entitled “Chronic lymphocytic leukemia
preceded by cold agglutinin disease: intraclonal immunoglobulin light-chain
diversity in VH4-34 expressing single leukemic B cells,” which appeared in
the November 1, 2002, issue of Blood (Volume 100:3419-3422), Laboratory
of Gene Expression, 1st Medical Faculty, Charles University, Prague, Czech
Republic, should have been included in the affiliations, and the grant
MSM/111100003 of the Ministry for Education, Youth and Sports of Czech
Republic should have been included in the acknowledgements.