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Plenary paper
Yolk sac–derived primitive erythroblasts enucleate during mammalian
embryogenesis
Paul D. Kingsley, Jeffrey Malik, Katherine A. Fantauzzo, and James Palis
The enucleated definitive erythrocytes of
mammals are unique in the animal kingdom. The observation that yolk sac–
derived primitive erythroid cells in mammals circulate as nucleated cells has led
to the conjecture that they are related to
the red cells of fish, amphibians, and
birds that remain nucleated throughout
their life span. In mice, primitive red cells
express both embryonic and adult hemoglobins, whereas definitive erythroblasts
accumulate only adult hemoglobins. We
investigated the terminal differentiation
of murine primitive red cells with use of
antibodies raised to embryonic ␤H1globin. Primitive erythroblasts progressively enucleate between embryonic days
12.5 and 16.5, generating mature primitive erythrocytes that are similar in size to
their nucleated counterparts. These
enucleated primitive erythrocytes circulate as late as 5 days after birth. The
enucleation of primitive red cells in the
mouse embryo has not previously been
well recognized because it coincides with
the emergence of exponentially expanding numbers of definitive erythrocytes
from the fetal liver. Our studies establish
a new paradigm in the understanding of
primitive erythropoiesis and support the
concept that primitive erythropoiesis in
mice shares many similarities with definitive erythropoiesis of mammals. (Blood.
2004;104:19-25)
© 2004 by The American Society of Hematology
Introduction
It was recognized more than 125 years ago that the mature red cells
of adult vertebrates circulate either in nucleated or enucleated
forms.1 The red cells of all birds, fish, reptiles, and amphibians
retain their nucleus and contain 3 filamentous systems: an actinspectrin–based membrane cytoskeleton, intermediate filaments that
attach the cytoskeleton to the nuclear membrane, and a group of
microtubules organized into a circumferential marginal band.2,3 In
contrast, the red cells of mammals lose intermediate filaments and
microtubules during terminal differentiation and enucleate prior to
entering the bloodstream. Thus, erythrocytes of adult mammals are
enucleated and contain only one filamentous system, a membrane
cytoskeleton.
Nearly 100 years ago, examination of mammalian embryos
revealed the presence of distinct nucleated and enucleated red
cells.4 The continuous circulation of small, enucleated red cells
during fetal and postnatal life was termed “definitive” erythropoiesis. Definitive erythropoiesis in the fetus is preceded by a
“primitive” erythroid program that is characterized by the transient
circulation of large, nucleated red cells that originate extraembryonically in the yolk sac.4,5 Because primitive erythroblasts in
mammals circulate as nucleated cells and are confined to the
embryo, they have been thought to share many characteristics with
the nucleated red cells of nonmammalian vertebrates when compared with the enucleated definitive red cells of fetal and adult
mammals.6,7
In the mouse embryo, primitive erythroid cells begin to develop
in yolk sac blood islands between embryonic days 7 and 8 (E7-8).8,9
With the onset of cardiac contractions at early somite pair stages
(E8.25), primitive erythroblasts enter the embryonic bloodstream10,11 where they remain until E16.5 when the primitive
lineage was thought to be extinguished.12,13 Definitive erythrocytes
begin to emerge from the fetal liver at E12.513,14 and rapidly
become the predominant cell type in the circulation. Definitive red
cells can be distinguished from their primitive counterparts by their
smaller size and by their accumulation of adult, but not embryonic,
hemoglobins.6,13,15 In contrast, primitive erythroblasts in the mouse
are large cells that accumulate both embryonic and adult
hemoglobins.15-17
More than 30 years ago, a population of enucleated red cells
with the same hemoglobin content as primitive erythroblasts was
described in the embryonic circulation of the mouse.14 Furthermore, large enucleated red cells have been noted in the bloodstream
of mouse embryos by several investigators,14,18-20 raising the
possibility that primitive erythroblasts might ultimately enucleate.
Studies in the Syrian hamster and in marsupials indicate that
primitive erythroblasts can enucleate,21-23 To determine whether
primitive erythroblasts enucleate in the mouse embryo and to
examine the transition from primitive to definitive erythropoiesis, we raised antibodies to embryonic ␤H1-globin and optimized the immunohistochemical identification of primitive
(␤H1-globin–positive) red cells. We report here that, contrary to
widely held opinion, murine primitive erythroblasts enucleate
and continue to circulate throughout late gestation and even into
the postnatal period. Our studies support the concept that the
From the Department of Pediatrics, Center for Human Genetics and Molecular
Pediatric Disease, University of Rochester Medical Center, Rochester, NY.
Reprints: James Palis, University of Rochester Medical Center, Department of
Pediatrics, Center for Human Genetics and Molecular Pediatric Disease, Box
703, 601 Elmwood Ave, Rochester, NY 14642; e-mail: james_palis@
urmc.rochester.edu.
Submitted December 4, 2003; accepted February 24, 2004. Prepublished
online as Blood First Edition Paper, March 18, 2004; DOI 10.1182/blood-200312-4162.
Supported by the National Institutes of Health (NIH) and the University of
Rochester School of Medicine and Dentistry.
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.
An Inside Blood analysis of this article appears in the front of this issue.
© 2004 by The American Society of Hematology
BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
19
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20
BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
KINGSLEY et al
primitive erythroid lineage in the murine embryo is truly
“mammalian” in nature.
Materials and methods
Collection of embryonic peripheral blood cells
Outbred Swiss Webster mice (Taconic, Germantown, NY) were mated
overnight, and vaginal plugs were checked the following morning (E0.3).
At specified times during gestation, mice were killed by cervical dislocation, and the uteri were removed from the peritoneum and washed with
several changes of phosphate-buffered saline (PBS). Embryos were dissected free of decidual tissues in a PB1 solution as described by Monk24 but
which was modified by removing penicillin and phenol red. After removal
of the placenta, individual, intact embryos were transferred to dishes
containing heparinized PB1 solution (as above with 12.5 ␮g/mL heparin).
Blood cells were collected by aspiration as they bled from severed
umbilical and vitelline vessels. Postnatal mice were killed by decapitation,
and peripheral blood cells were collected from jugular vessels into
heparinized PB1 solution. Cytospins were prepared with 50 000 cells spun
at 400 rpm for 3 minutes (Cytospin2; Thermo Shandon, Pittsburgh, PA) and
stained with May-Grünwald-Giemsa (Sigma Diagnostics, St Louis, MO),
and photographed with a Nikon Optiphot microscope equipped with a ⫻ 40
objective (NA 0.85) and a SPOT RT-slider digital camera (Diagnostic
Instruments, Sterling Heights, MI). Images were processed in Photoshop
(Adobe, San Jose, CA).
Total red cell counts in timed E12.5, E14.5, and E16.5 embryos
At least 3 individual conceptuses from each timed pregnancy were carefully
dissected free of decidual tissues and transferred intact with their placentas
into individual dishes of modified PB1. The yolk sac and umbilical vessels
were severed at their attachment to the placenta using no. 5 watchmaker’s
forceps, and the embryos were immediately transferred into fresh 35-mm
dishes of heparinized PB1 solution. Each embryo was allowed to exsanguinate from severed umbilical, vitelline, and jugular vessels, and all blood
cells in the dish were collected and counted by hemacytometer. Data from
a minimum of 3 different timed pregnancies were obtained for each
time point.
Coulter analysis of peripheral blood cells
To quantify the size of the circulating red cells, samples of peripheral blood
were diluted into PBS medium and analyzed on a Coulter ZM with
channelyzer with preset gain of 4 and attenuation of 16 (Coulter Electronics, Essex, England).
Raising of anti–␤H1-globin and anti–␤major-globin antibodies
Peptide sequences of the murine ␤H1-globin and ␤major-globin proteins
were compared to identify regions of divergence. Rabbit polyclonal
antipeptide antibodies were generated to amino acids 71 to 84 of accession
number NP_032245 (␤H1-globin) and the corresponding region of ␤-major
globin (accession no. P02088) by Biosynthesis (Lewisville, TX). Both
primary antisera and Protein A–purified immunoglobulin G (IgG) fractions
(CPG, Lincoln Park, NJ; prepared according to manufacturer’s instructions)
provided reproducible results on paraformaldehyde-fixed tissues and ethanolfixed blood cells.
Immunohistochemical analysis
E10.5 and E12.5 mouse embryos were dissected free of maternal tissues,
fixed overnight in fresh 4% buffered paraformaldehyde, embedded in
paraffin, and sectioned. Immunohistochemistry was performed using standard avidin-biotin complex (ABC) protocols (Vector Laboratories, Burlingham, CA) after antigen retrieval by pretreatment of the slides for 15 minutes
at 100°C in 0.1M Tris (tris(hydroxymethyl)aminomethane), pH 6.
To identify blood cells containing ␤H1-globin by immunohistochemistry, it was necessary to develop a protocol that optimized cell morphology,
globin retention, and antigenicity, as well as cell dispersal that allowed for
semiautomated morphometric analysis. Blood cells collected from timed
conceptuses were centrifuged at 1000g for 5 minutes and resuspended at
approximately 50 000 cells/␮L in 83% modified PB1, 12.5 ␮g/mL heparin,
3.67% bovine serum albumin (BSA; Sigma, St Louis, MO). Diluted cells (3
␮L) were smeared onto untreated glass slides using standard wedge
technique. After air drying, smears were fixed with 100% ethanol for 5
minutes and stored at room temperature for up to 2 months. Following
pretreatment in 3% Triton X-100 in PBS for 6 to 14 hours, immunohistochemistry was performed according to instructions included with Vector
Laboratories’ ABC kit with alkaline phosphatase and Vector Red as the
enzyme and substrate, respectively. No specific cross-reaction was observed with preimmune sera or with preimmune IgG. For the experiment in
Figure 2D, cells were incubated with DAPI (4⬘,6-Diamidino-2-phenylindole dihydrochloride) at 2 ␮g/mL in PBS for 10 minutes, after immunohistochemistry, then mounted in aqueous mounting medium. Cells were
photographed in fluorescence with either a Nikon Otiphot microscope
equipped with a ⫻ 4 objective (NA 0.13) (Figure 2A), ⫻ 10 objective (NA
0.50) (Figure 2B-C), ⫻ 40 objective (NA 0.85) (Figure 2G) with paired
phase-contrast optics, or with a Nikon Eclipse TE 2000-S microscope
equipped with a ⫻ 40 objective (NA 0.60) (Figure 2D-E) with paired
Hoffman modulation contrast optics and a SPOT RT-Slider camera. Images
were processed in Photoshop.
Morphometric analysis
To determine the areas of peripheral blood cells, smears of E15.5 blood
were analyzed for ␤H1-globin expression and photographed using the
SPOT RT-slider digital microscope camera. These images were processed
in Photoshop (Adobe Systems, San Jose, CA) with Fovea Pro quantitative
image analysis plug-ins (Reindeer Graphics, Asheville, NC).
Results
Changes in the distribution of red cell populations in the
embryonic circulation
The first red cells in the mouse embryo arise in blood islands of the
yolk sac and consist exclusively of large primitive erythroblasts.
Examination of smears of peripheral blood cells indicates that
nucleated primitive erythroblasts constitute the only circulating red
cell population at E8.5, E9.5, and E10.5 (data not shown). These
cells undergo progressive maturation, transitioning from proerythroblasts at E8.5 to polychromatophilic erythroblasts by E11.5
(Figure 1B and data not shown). These primitive erythroblasts are
extremely large cells with volumes ranging from 300 fl to 750 fl
(Figure 1A). This volume is in marked contrast to adult erythrocytes that have a mean cell volume (MCV) of approximately 70 fl
(Figure 1A).
Between E11.5 and E12.5, a second population of red cells
emerges in the circulation (Figure 1C, arrow). This population of
cells has an MCV of 150 fl, which is intermediate in size when
compared with primitive erythroblasts and adult red cells. The
appearance of this second cell population coincides with the
presence of smaller, enucleated red cells on peripheral smears
(Figure 1D, arrow). These fetal liver–derived definitive erythrocytes rapidly become the predominant cell population between
E14.5 and E15.5, as evidenced by the Coulter analysis (Figure
1E,G). Closer examination of peripheral blood smears from E14.5
embryos revealed a population of large enucleated red cells (Figure
1H, arrowheads). These large erythrocytes were evident as early as
E12.5, (Figure 1D, arrowhead) and were similar in size to
nucleated primitive red cells, raising the possibility that they are
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BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
PRIMITIVE MAMMALIAN ERYTHROBLASTS ENUCLEATE
21
primitive erythroblasts that contain both embryonic (␤H1) globin
and a nucleus, as confirmed by DAPI staining (Figure 2D-E, black
arrows). The second population consisted of definitive erythrocytes
that lack both ␤H1-globin expression and a nucleus (Figure 2D-E,
blue arrows). The third population consisted of cells that contain
␤H1-globin but lack a nucleus (Figure 2D-E, red arrows). The
expression of embryonic globin by this third population of
erythrocytes is consistent with their derivation from the primitive
erythroid lineage.
As primitive red cells are significantly larger than definitive red
cells (Figure 1), we compared the relative size of these 3 cell
populations by quantifying cell areas. As shown in Figure 2F, the
nucleated and enucleated ␤H1-globin–positive cell populations
(black square and red diamond, respectively) have similar cell
areas and each is significantly larger than the ␤H1-gobin–negative
red cell population (blue circle). We thus identified a population of
enucleated red cells in the bloodstream of the mouse embryo that
shares both embryonic globin expression and cell size with
primitive erythroblasts. These results indicate that yolk sac–
derived primitive erythroblasts can enucleate and circulate as
erythrocytes.
Changes in red cell population dynamics between E10.5 and
E17.5 of mouse gestation
Figure 1. Changes in the cellular composition of peripheral blood between
E11.5 and E14.5 of mouse gestation. (A,C,E,G) Cell size distribution of circulating
embryonic blood cells (black area). An independent sample of adult murine red cells
(gray area) is superimposed on each Coulter analysis. The x-axis represents cell
volume (femtoliters); y-axis, relative cell number. (B,D,F,H) Peripheral blood cells
stained with May-Grünwald-Giemsa. Some of the small and large enucleated red
cells are marked with arrows and arrowheads, respectively. The scale bar represents
10 ␮m.
derived from the primitive erythroid lineage. To test this hypothesis, antibodies were raised to embryonic globin to specifically
distinguish primitive red cells from other circulating blood cells.
The specificity of the anti–␤H1-globin antibodies was tested by
immunohistochemical analysis of sections of E10.5 and E12.5
embryos. As shown in Figure 2A, anti–␤H1-globin antibodies
specifically bind to circulating E10.5 primitive erythroblasts but do
not bind to adjacent mesodermal (m) or endodermal (e) tissues. At
E12.5, anti–␤H1-globin antibodies decorate primitive red cells but
do not bind to nucleated definitive erythroblasts within the liver
(Figure 2B). The ␤H1-globin–positive cells in the fetal liver are
primitive red cells localized within blood vessels. In contrast,
antibodies raised to the adult ␤major-globin protein decorated both
circulating primitive erythroblasts and differentiating definitive
erythroblasts in the fetal liver (Figure 2C). No ␤H1-globin–positive
cells were detected in adult peripheral blood (data not shown).
These results confirm that the anti–␤H1-globin antibodies recognize yolk sac–derived primitive red cells but not fetal or adult
definitive erythroid cells.
Identification of enucleated primitive red cells
To determine whether primitive erythroid cells might enucleate, we
established conditions for ␤H1-antibody staining of smeared
circulating blood cells. Three different cell populations were
identified when we examined peripheral blood cells from E13.5
and E15.5 fetuses (Figure 2D-E). The first population consisted of
As has been noted previously25 and is evidenced by progressive
changes in cell morphology (Figure 1), primitive erythroid cells
differentiate in a semisynchronous manner. Our observation that
primitive erythroid cells can enucleate raised the possibility that
enucleation is a normal component of primitive erythropoiesis, as
this lineage terminally differentiates in the bloodstream. Alternatively, these enucleated forms could be rare cells that deviate from
an inherent nonmammalian character of the primitive erythroid
lineage in mice. To distinguish between these alternatives, we
systematically analyzed the cellular composition of peripheral
blood between E11.5 and E17.5 of mouse gestation. Examples of
this immunohistochemical analysis are shown in Figure 2G.
Examination of peripheral blood smears following immunohistochemistry for ␤H1-globin revealed that all of the primitive red
cells at E10.5 and E11.5 were nucleated (Figure 2G, Figure 3A,
white column). We first detected small numbers of enucleated
␤H1-globin–positive primitive erythrocytes at E12.5 (Figure 3A,
black column). There is a progressive transition to enucleated cells
that is completed by E17.5 (Figures 2G and 3A). This transition
from nucleated to enucleated forms between E12.5 and E17.5
supports the concept that primitive erythroid cells terminally
differentiate in the bloodstream and ultimately become enucleated
primitive erythrocytes. Our immunohistochemical analysis also
allowed us to examine the presence of definitive red cells in the
bloodstream during the second half of mouse gestation. Enucleated
definitive (␤H1-globin–negative) erythrocytes were first detected
at E11.5 and continue to be a minor component of the circulation at
E12.5 (Figure 3B, light gray column). However, by E13.5,
definitive red cells constitute more than 50% of the circulating
blood cells. This transition continues so that by E17.5, 99% of the
circulating cells are definitive erythrocytes (Figures 2G and 3B).
We also noted a minor population of nucleated ␤H1-globin–
negative cells (Figure 3B, clear columns) that likely comprise a
diverse group of cells, including myeloid cells, definitive erythroblasts released from the fetal liver prior to enucleation, and
contaminating nonhematopoietic cells. They were most prevalent
at E13.5 and always constituted less than 5% of the peripheral
blood cells.
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22
KINGSLEY et al
BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
Figure 2. Immunohistochemistry with antiglobin antibodies. (A) E10.5 yolk sac stained with anti–␤H1-globin antibody. Positive primitive erythroblasts are evident
throughout a large blood vessel, whereas adjacent mesoderm-derived (m) and endoderm (en) cells are negative. The scale bar represents 25 microns. (B-C) E12.5
neighboring embryo sections stained with anti–␤H1-globin (B) and anti–␤major-globin (C) antibodies. The anti–␤H1-globin antibodies decorate primitive erythroblasts in blood
vessels (white arrows). Anti–␤major-globin antibodies decorate both primitive red cells in vessels and definitive erythroblasts maturing in the liver (liver). Int indicates intestine.
The scale bar represents 50 ␮m. (D) E13.5 peripheral blood cells stained both with anti–␤H1-globin antibodies and with DAPI (left). A paired Hoffman modulation contrast
image (HMC, right) shows the ␤H1-globin–positive nucleated cells (black arrows), ␤H1-globin–negative enucleated cells (blue arrows), and ␤H1-globin–positive enucleated
cells (red arrows). The scale bar represents 10 ␮m. (E) E15.5 peripheral blood cells stained with anti–␤H1-globin antibodies (left). A paired Hoffman modulation contrast image
(HMC, right) of the same field of cells indicates a ␤H1-globin–positive nucleated cell (black arrow), the ␤H1-globin–negative enucleated cells (blue arrows), and a
␤H1-globin–positive enucleated cell (red arrow). The scale bar represents 25 ␮m. (F) Morphometric analysis of cell area of 3 populations of peripheral blood cells at E15.5 of
mouse gestation. Nucleated (black square) and enucleated (red diamond) ␤H1-globin–positive cells are both similar in size and are significantly larger than
␤H1-globin–negative definitive erythrocytes (blue circle). Ordinate units are ␮m2. (G) Immunohistochemical analysis of ␤H1-globin expression in circulating blood cells from
E11.5, E14.5, and E17.5 mouse embryos (right column) with paired phase contrast images (left column). At E14.5, both nucleated and enucleated (arrow) ␤H1-globin–positive
cells are evident. All images are at the same magnification. Scale bar represents 10 ␮m.
Our analysis indicates that 2 major transitions occur between
E12.5 and E17.5 of gestation. The first is progressive transition
from nucleated to enucleated primitive red cells (Figure 3A). The
second is a rapid transition from primitive to definitive red cells
(Figure 3B). These findings suggest that the process of enucleation
leads to the demise of primitive red cells. If this were the case, we
would expect the total number of primitive red cells to decrease
significantly as they enucleate. Alternatively, the primitive erythroid lineage could persist in the circulation but be diluted by the
influx of definitive erythrocytes from the liver. To distinguish
between these alternatives, we estimated the total number of
primitive and definitive red cells in the embryo between E12.5,
when enucleation begins, and E16.5, when more than 90% of the
primitive red cells have enucleated. At E12.5, the embryo contains
approximately 5 million red cells, almost all of which are primitive
(Table 1). Total red cell numbers expand 30-fold over the next 4
days. This massive increase in red cell mass is due almost entirely
to the entry of definitive red cells from the fetal liver into the
circulation. The number of primitive red cells increases only
marginally between E12.5 and E16.5 (Table 1). Because primitive
red cells cease dividing by E13.5,26,27 these results indicate that the
process of enucleation is not associated with loss of primitive red
Figure 3. Changes in the distribution of circulating red cell
populations between E10.5 and E17.5 of mouse gestation.
(A) Changes in the percentage of nucleated (white columns) and
enucleated (black columns) ␤H1-globin–positive primitive red
cells in the bloodstream. There is a progressive transition from
nucleated to enucleated forms. (B) Changes in the distribution of
␤H1-globin–positive nucleated (white columns) and enucleated
(black columns) cells and in ␤H1-globin–negative nucleated
(clear columns) and enucleated (gray columns) cells. Primitive
red cells become a progressively smaller component of the
circulation between E12.5 and E17.5. Results in this figure are
derived from the examination of 14 855 cells from more than 60
fetuses from 22 timed litters.
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BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
PRIMITIVE MAMMALIAN ERYTHROBLASTS ENUCLEATE
Table 1. Total number ⴞ SEM of circulating red blood cells (RBCs)
and of primitive red cells in E12.5, E14.5, and E16.5 mouse embryos
Total RBCs,
ⴛ 106 per embryo
Primitive RBCs,
ⴛ 106 per embryo
Nucleated
primitive RBCs, %
n
E12.5
5.3 ⫾ 0.9
4.8 ⫾ 0.6
91.2 ⫾ 4.3
3
E14.5
26.3 ⫾ 6.9
6.0 ⫾ 2.2
22.6 ⫾ 9.4
3
E16.5
151.7 ⫾ 28.6
10.8 ⫾ 4.5
6.5 ⫾ 2.0
4
There are no significant changes in the numbers of primitive red cells as they
enucleate between E12.5 and E16.5. n indicates the number of independent
experiments.
cells from the circulation. Rather, the disappearance of nucleated
primitive red cells in the bloodstream is due both to their
progressive enucleation and to their dilution by increasing numbers
of definitive erythrocytes from the fetal liver.
Primitive erythrocytes continue to circulate after birth
Our results indicate that the primitive erythroid lineage is not
extinguished as previously thought when nucleated red cells are no
longer present in the embryonic bloodstream.12,13 To determine
whether primitive red cells circulate beyond E17.5, we analyzed
peripheral blood from late gestation mouse fetuses and postnatal
pups for the presence of ␤H1-globin–positive red cells. Primitive
erythrocytes were evident at E18.5, at birth (E19.5), and for several
days after birth (data not shown). Even at postnatal day 5, rare
␤H1-positive red cells were detected in 1 of 3 mice examined.
Because nucleated primitive red cells are not evident after day 16.5,
these results indicate that primitive red cells can circulate as
erythrocytes for several days after enucleating.
Discussion
Primitive erythroid cells are necessary for survival of the mammalian embryo because they comprise a critical component of a
functional circulatory system.28 Primitive erythroid cells originate
in blood islands of the yolk sac and expand exponentially in
numbers between E8.5 and E10.5, when robust circulation is
reflected by the high proportion of the embryo’s cell mass that is
red blood cells.10 As exemplified by the disruption the scl and
GATA-1 genes, the lack of primitive red cells leads to fetal death in
the mouse between E9.5 and 10.5.29-31 The primitive erythroid
lineage continues to expand in numbers until E12.5 to 13.5 when
cell division ceases. Because primitive red cells constitute the
predominant cell type in the circulation through E12.5 (Figure 3B),
anemia in the embryo before E13.5 reflects a disorder of the
differentiation or circulation of the primitive erythroid lineage.
After this time, the growing fetus’s need for an increasing red cell
mass is met by the exponential expansion of the definitive erythroid
lineage. The 30-fold increase in red cell number between E12.5 and
E16.5 is due almost entirely to the emergence of definitive
erythrocytes from the fetal liver. In the complete absence of
definitive red cells, as exemplified by mice lacking c-myb, the
primitive erythroid lineage can sustain fetal survival until E15.5
to E16.5.32
Previous investigators have used cell size,18,33 acid elution,19,34,35 hemoglobin content,14 and antibodies generated to
embryonic hemoglobins13,36 to identify primitive red cells in the
rodent embryo. Because ␤H1-globin is restricted to the primitive
erythroid lineage of the mouse, we generated antibodies to a
peptide corresponding to an unique region of the ␤H1-globin chain
23
and optimized an immunohistochemical protocol to identify primitive red cells. These experimental data reported here indicate that
primitive erythroblasts normally enucleate during terminal differentiation. It is likely that enucleated primitive erythrocytes have not
previously been well recognized in the mouse embryo because
their appearance coincides with the massive entry into the bloodstream of enucleated definitive erythrocytes from the liver.
Our direct measure of cell volume with use of the Coulter
counter indicates that E9.5 to E12.5 primitive erythroblasts in mice
are extremely large cells with volumes ranging between 300 and
800 fl. This wide range of cell volumes likely reflects the presence
of dividing cells, as evidenced by the exponential expansion in red
cell numbers10 and the presence of mitotic figures at E8.5 to E11.5
(data not shown). By E13.5, when cell division has ceased, the
distribution of primitive red cell size has become bell-shaped, and
the mean cell volume of primitive erythroid cells is approximately
400 fl (Figure 1E). These findings are consistent with those
previously reported for E13 to E15 primitive erythroblasts.37 Yolk
sac–derived primitive red cells are approximately 3-fold larger than
fetal liver–derived definitive erythrocytes and 5- to 6-fold larger
than adult murine erythrocytes. Although the mechanisms responsible for the differences in cell size among these different red cell
lineages are not known, they may relate to differential rates of cell
division. Steiner38 has determined that primitive erythroblasts
divide more slowly than fetal liver–derived definitive erythroblasts,
allowing for increased hemoglobin accumulation during terminal
maturation.
Our studies establish a new paradigm in the understanding of
the primitive erythroid lineage and indicate that primitive erythropoiesis in mammals shares many processes with its definitive
counterpart, including progressive phases of (1) lineage-committed
progenitors, (2) erythroblast maturation with enucleation, and (3)
circulation as mature erythrocytes (Figure 4). Although definitive
red cells arise from committed BFU-E and CFU-E progenitors,
primitive red cells arise from an unique EryP-CFC progenitor.39 We
have previously identified this first phase of committed progenitors
for the primitive erythroid lineage in the mouse embryo.17 EryPCFCs arise during early gastrulation (E7.25) and rapidly expand in
numbers within the yolk sac. However, this wave of primitive
erythroid progenitors is transient, lasting only 48 hours before
being extinguished.17,36
Figure 4. Primitive and definitive erythropoiesis each share 3 phases of
differentiation. Committed erythroid progenitors give rise to maturing nucleated
erythroblasts that ultimately generate enucleated erythrocytes. The specific erythroid
progenitors are EryP-CFCs (primitive erythroid colony-forming cells), BFU-Es (erythroid burst-forming units), and CFU-Es (erythroid colony-forming units). The characteristics common to primitive and definitive erythroblast maturation in mammals are
listed along with references pertaining to primitive erythropoiesis in the mouse. We
propose that a major distinction between primitive and definitive erythropoiesis in
mammals is that primitive red cells differentiate while circulating in the bloodstream,
whereas definitive red cells enter the circulation only after completing their maturation
extravascularly.
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BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
KINGSLEY et al
The second phase of erythropoiesis consists of erythroblast
maturation (Figure 4). In the definitive erythroid lineage this phase
is characterized by several cell divisions and a progression of
morphologically identifiable forms, resulting from the accumulation of hemoglobin in the cytoplasm and condensation of the
nucleus.40 With the loss of vimentin intermediate filaments, the
nucleus begins to move within the cytoplasm and is eventually
discarded.41 Similar events occur in primitive erythroid cells as
they circulate in the embryonic bloodstream. They continue to
divide26,27 and accumulate hemoglobin until E13.5.14 They mature
from proerythroblasts at E8.5 to orthochromatic erythroblasts at
E12.5 to E15.5 (Figure 1; and data not shown). Their nuclei
progressively condense (Figure 1) and begin to move within the
cell coincident with the loss of vimentin intermediate filaments.27
Here, we show that murine primitive erythroblasts, like their
definitive counterparts, ultimately enucleate during terminal
differentiation.
The third phase of definitive erythropoiesis in mammals is
characterized by the circulation of enucleated erythrocytes (Figure
4). The maintenance of primitive red cell numbers between E12.5
and E16.5 suggests that enucleating primitive erythroblasts are not
lost from the circulation. Primitive red cells are still present in the
bloodstream as late as 5 days after birth, indicating that primitive
erythrocytes circulate for several days after enucleating. These
findings support the concept that enucleation is a normal developmental process during primitive erythroid differentiation. It is
likely that this third phase of primitive erythropoiesis has not been
recognized because primitive red cells are relegated to a minor
component of the red cell mass by the massive influx of definitive
red cells during late gestation (Figure 3B).
The mature red cells of nonmammalian vertebrates are distinguished from mammalian red cells, not only by the retention of a
nucleus, but also by the presence of marginal bands and intermediate filaments. In contrast, definitive erythrocytes of almost all
mammals, with the exception of the immature red cells of camels,42
lack these structural features. Although marginal bands have been
detected in the primitive red cells of marsupials,23 it is not clear
whether true circumferential marginal bands exist in the primitive
erythroblasts of mice.43,44 Consistent with their enucleation, murine
primitive erythroblasts lose intermediate filaments during their
maturation in the bloodstream.27 These features further support the
concept that the primitive erythroid lineage in mice shares many
similarities with the definitive erythroid lineages of mammals when
compared with the red cell lineages of nonmammalian vertebrates.
Although primitive and definitive erythropoiesis in mammals
share many characteristics, they are distinguished by differences in
transcriptional regulation (eg, c-myb), cell size, and types of
hemoglobin accumulated. We conclude that another distinguishing
feature of primitive erythropoiesis in mice is not the retention of a
nucleus, but rather that primitive red cells mature in the bloodstream (Figure 4). This is in striking contrast to definitive erythroblasts that mature extravascularly within erythroblast islands in
close association with macrophage cells and stromal elements.45,46
The mechanisms that regulate primitive erythroid maturation in the
absence of stromal interactions are not currently known. The ability
to obtain circulating primitive erythroblasts at progressive stages of
maturation will aid in better understanding the molecular underpinnings of mammalian red blood cell maturation and the mechanisms
directing enucleation.
Acknowledgments
We thank Peter Keng of the University of Rochester Cancer Center
for his kind assistance with the Coulter Counter analysis and Anne
Koniski for exemplary animal husbandry. We also thank members
of the laboratory, as well as Bill Cohen and Rick Waugh, for helpful
discussions and critical comments.
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2004 104: 19-25
doi:10.1182/blood-2003-12-4162 originally published online
March 18, 2004
Yolk sac−derived primitive erythroblasts enucleate during mammalian
embryogenesis
Paul D. Kingsley, Jeffrey Malik, Katherine A. Fantauzzo and James Palis
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