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Review article
The embryonic origins of erythropoiesis in mammals
Margaret H. Baron,1-5 Joan Isern,1,4 and Stuart T. Fraser1,4,5
Departments of 1Medicine, 2Developmental and Regenerative Biology, and 3Oncological Sciences, 4Tisch Cancer Institute, and 5Black Family Stem Cell
Institute, Mount Sinai School of Medicine, New York, NY
Erythroid (red blood) cells are the first cell
type to be specified in the postimplantation
mammalian embryo and serve highly specialized, essential functions throughout gestation and postnatal life. The existence of
2 developmentally and morphologically distinct erythroid lineages, primitive (embryonic) and definitive (adult), was described
for the mammalian embryo more than a
century ago. Cells of the primitive erythroid
lineage support the transition from rapidly
growing embryo to fetus, whereas definitive
erythrocytes function during the transition
from fetal life to birth and continue to be
crucial for a variety of normal physiologic
processes. Over the past few years, it has
become apparent that the ontogeny and
maturation of these lineages are more com-
plex than previously appreciated. In this
review, we highlight some common and
distinguishing features of the red blood cell
lineages and summarize advances in our
understanding of how these cells develop
and differentiate throughout mammalian ontogeny. (Blood. 2012;119(21):4828-4837)
Introduction
During embryonic development, hematopoiesis functions in the rapid
production of large numbers of erythroid cells that support the growth
and survival of the embryo/fetus and, later, in the generation of a pool of
hematopoietic stem cells (HSCs) that persist throughout the life of the
adult animal.1,2 Hematopoiesis in the developing vertebrate embryo
occurs in multiple waves and in several different anatomic sites (Figure
1). The first wave occurs in the yolk sac in both mouse and humans3-5
and produces primarily primitive erythroid cells (EryP) as well as
macrophages and megakaryocytes.3,6 The second wave also arises in the
yolk sac but is “definitive,” composing erythroid, megakaryocyte, and
several myeloid lineages.7,8 The third wave emerges from HSCs
produced within the major arteries of the embryo, yolk sac, and
placenta.1-3,9 HSCs home to and expand within the fetal liver and
eventually seed the bone marrow.10,11 Definitive erythroid cells are
produced continuously from HSCs in the bone marrow throughout
postnatal life.1,2
Before the initiation of definitive (adult-type) hematopoiesis
from multipotent stem cells in the fetal liver, the embryonic
circulation is dominated by large, nucleated erythroid cells of the
EryP lineage. Primitive erythropoiesis is transient: EryP progenitors are produced in the yolk sac of the embryo for only a brief
period (⬃ 2 days).7,12 Their terminally differentiated progeny
persist in the circulation through the end of gestation and even for a
while after birth.13 However, they are rapidly outnumbered by the
rapidly expanding population of definitive erythroid cells (EryD)
arising from the growing fetal liver.13,14
Failure in primitive erythropoiesis (as observed, for example,
after targeted disruption of genes encoding the transcription factors
Gata-1, Gata-2, Lmo2, or Scl) is uniformly associated with embryonic
lethality.1,15 The importance of this lineage is underscored by the fact
that primitive erythropoiesis is conserved among vertebrates.1,15
Emergence of primitive erythroid cells in the
yolk sac
During gastrulation, a single epithelial cell layer (the epiblast) is
transformed into the 3 germ layers of the embryo (ectoderm,
Submitted January 1, 2012; accepted February 9, 2012. Prepublished online
as Blood First Edition paper, February 15, 2012; DOI 10.1182/blood-2012-01153486.
4828
mesoderm, and endoderm), and the basic body plan of the animal is
established.16 In the mouse, gastrulation initiates around embryonic
day (E) 6.5 (Figure 1A), when surface ectoderm cells undergo an
epithelial-mesenchymal transition and become migratory, moving
through the primitive streak and into the extraembryonic region of
the embryo. There they form a mesodermal layer that lies directly
adjacent to the visceral endoderm, in the early yolk sac.4 In the
mouse, EryP are first detected at around E7.5, within “blood
islands”5 (Figure 1A). They arise from mesodermal progenitors
with restricted hematopoietic potential.
A role for yolk sac (visceral) endoderm in hematopoietic and
vascular induction was suggested by classic studies in the chick
embryo and was supported by the observation that embryoid bodies
formed from Gata4-deficient embryonic stem (ES) cells lacked
visceral endoderm and showed greatly reduced primitive erythropoiesis.17 Explant experiments with mouse embryos suggested that
soluble signals from visceral endoderm activate hematopoiesis and
vascular development during gastrulation17 and function, at least in
part, through Indian hedgehog (Ihh), which may in turn regulate
expression of BMP4 from the adjacent mesoderm.18,19 Recent
coculture experiments have indicated that signals from primitive
endoderm-derived extraembryonic endoderm cells20 can stimulate
the expansion of EryP progenitors.21 Candidate-secreted factors
identified on the basis of a microarray analysis include vascular
endothelial growth factor and Ihh.21
Wnt/␤-catenin signaling regulates specification of the EryP
lineage from mesoderm in differentiating mouse ES cells22 in a
process that requires inhibition of the Notch pathway.23 A microarray analysis of maturing primary mouse EryP isolated at distinct
developmental stages indicated that a number of Wnt/␤-catenin
pathway genes are expressed in EryP progenitors and are rapidly
down-regulated as the cells mature.12 That the Wnt pathway is
active and functions autonomously in EryP progenitors was
suggested by immunostaining for activated (stabilized) ␤-catenin
and by fluorescence of a nuclear-GFP Wnt/␤-catenin reporter in the
blood islands of the yolk sac.12
© 2012 by The American Society of Hematology
BLOOD, 24 MAY 2012 䡠 VOLUME 119, NUMBER 21
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Figure 1. Shifts in site of hematopoiesis during
mouse and human development. (A) Hematopoietic
development in the mouse. Formation of mesoderm
during gastrulation (around E6.5), development of yolk
sac blood islands (⬃ E7.5), emergence of HSCs in the
aorta-gonad-mesonephros region (E10.5; other sites such
as large arteries and placenta not shown), active fetal
liver hematopoiesis (E14.5), and hematopoiesis in the
bone marrow of the late gestation fetus (E18.5) and adult
animal. Active circulation begins at approximately E9.0.43
(B) Hematopoietic development in the human embryo.
An embryo at yolk sac stage of hematopoiesis (day 17),
at the time of the first hepatic colonization by HSCs (day
23), arterial cluster formation (day 27), second hepatic
colonization (day 30), and bone marrow colonization
(10.5 weeks). Active circulation begins at approximately
day 21.3
The close spatial and temporal association between primitive
erythropoiesis and vasculogenesis (the formation of blood vessels
from angioblasts) in the yolk sac led to the concept of a common
progenitor termed the “hemangioblast.”1,15 Hemangioblasts were
thought to give rise to “blood islands,” clusters of EryP surrounded
by endothelial cells within the mesodermal layer of the yolk sac.5
Experimental support for the hemangioblast came from studies of
differentiating mouse and human ES cells24,25 and from mouse
embryos.26 Although “blast colony”-forming cells (BL-CFCs) were
hypothesized to be the in vitro equivalent of the hemangioblast,24
the majority of BL-CFC activity in the mouse embryo was found
not in the yolk sac but in the posterior primitive streak.26 BL-CFCs
are not strictly bipotential: in addition to primitive and definitive
hematopoietic cell types,6,24 they can also give rise to smooth
muscle in vitro.27
On the basis of lineage-tracing experiments, it was concluded
that EryP and angioblasts in the yolk sac do not share a common
progenitor.28 Analysis of mouse tetrachimeras expressing different
fluorescent proteins has suggested that blood islands are polyclonal
and, therefore, that hematopoietic and angioblastic lineage commitment in the yolk sac does not involve a 2-potential progenitor.29
However, it may be possible to reconcile these findings with the
BL-CFC analyses from mouse embryos26: the rare hemangioblasts
in the posterior streak may produce more restricted progenitors (eg,
megakaryocyte-erythroid progenitors, EryP progenitors, angioblasts) that then colonize the yolk sac. In a careful immunohistochemical analysis of the early mouse yolk sac,5 independent blood
islands could not be identified. Instead, a “blood band” of
CD41-positive hematopoietic progenitor cells was observed
(⬃ E7.75) to gradually circumscribe the proximal yolk sac.5 It was
proposed that this blood band, containing primitive erythroid cells
and more sparsely distributed endothelial cells, is later subdivided
by layers of endothelial cells.5 Consistent with this idea, 2 waves of
angioblast development have been identified in the yolk sac: one
closely associated with and a second independent of hematopoiesis.30 Therefore, it remains unclear whether an equivalent of the
hemangioblast exists in vivo.
Although it has been proposed that an Flk1-positive hemangioblast undergoes a transition to a “hemogenic” endothelial intermediate before the emergence of hematopoietic cells,31,32 the early
wave of blood development in the yolk sac does not progress
through a frank endothelial intermediate.5,33
Primitive erythropoiesis
Embryonic and fetal erythropoiesis has been best studied in the
mouse. Primitive and definitive erythroid cells are distinct lineages
that arise from different populations of mesoderm (posterior and
lateral plate, respectively) generated during gastrulation.4 Cells of
the 2 erythroid lineages differ in a number of important characteristics. The “megaloblastic” EryP are much larger than either fetal
liver or adult EryD (Figure 2), express distinct globin genes,34 and
differ in their O2-carrying capacity and response to low oxygen
tension.35 Whereas EryP form only in the yolk sac, progenitors for
EryD are found both in the yolk sac and fetal liver.7,36 The primitive
and definitive erythroid lineages also differ in their dependence on
specific cytokines, transcription factors, and downstream regulatory pathways.
More than a century ago, it was observed that mammalian blood
contains distinct nucleated and enucleated erythroid cells, leading
to the conventional wisdom that a key distinguishing feature of
primitive and definitive erythroid cells at all stages was the
presence or absence of a nucleus.37 In contrast with EryD, which
enucleate extravascularly in the fetal liver or adult bone marrow
before entering the bloodstream, EryP were thought to retain their
nuclei throughout their development. These large circulating cells
were thought to represent a “primitive” form of erythropoiesis
because, like the nucleated red cells of nonmammalian vertebrates,
they formed in the yolk sac and were confined to embryonic stages
Figure 2. Primitive red blood cells are megaloblastic. EryP (E10.5) were mixed
with (A) fetal (E17.5) or (B) maternal peripheral blood erythrocytes, cytospun, and
stained with Giemsa as described by Fraser et al.13 Scale bar represents 20 ␮m.
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4830
BARON et al
BLOOD, 24 MAY 2012 䡠 VOLUME 119, NUMBER 21
specifically drive expression of reporters, including lacZ,17,18 a
pan-cellular green fluorescent protein (GFP),13,18 and a histone
H2B-GFP fusion,42 within the mouse EryP compartment of transgenic mice (Figure 5). The fluorescent reporters have made it
possible to isolate EryP progenitors12 and maturing EryP,13,42 to
track EryP enucleation,13,42 and to generate a genome-wide transcriptome of this lineage at distinct stages of its development.12
Primitive erythroid progenitors
Figure 3. Cytologic changes during primitive erythroid maturation. Giemsastained cytospin preparations of FACS-sorted E8.5 EryP from dispersed ␧-globin-H2BGFP embryos12,42 or peripheral blood from wild-type embryos at E9.5 to E14.5.13
Panels E9.5 to 14.5 were taken from Fraser et al.13 Scale bar represents 20 ␮m.
of development. However, it is now clear that primitive erythroblasts in the mouse embryo are nucleated for only part of their life
span, maturing and expelling their nuclei around midgestation.13,14
They then continue to circulate as enucleated cells until and
perhaps, for a short time, past the time of birth.13 In addition to
enucleation late in their maturation,13,14 the 2 lineages share a
number of other features, including terminal differentiation from
unipotential progenitors, progressive stages of maturation as nucleated erythroblasts,13,14,38 accumulation of hemoglobin, decrease in
cell size, nuclear condensation, and regulation by erythroid transcription factors, such as GATA1 and EKLF/KLF1.12-14,37,39 The cytologic changes observed during primitive erythroid maturation are
shown in Figure 3. EryP display “maturational” gene switching
within the ␤-globin locus40 involving sequential changes in expression from ␤h1- to εY- (both embryonic) to adult ␤maj- (␤1) globin.
It is evident from Figure 4A that εY-globin remains the predominant transcript throughout EryP development and that, even at late
times, ␤maj-globin expression remains low. The data are presented
in a normalized form in Figure 4B, with peak expression set at
100 for each gene, to emphasize the sequential gene expression.
Primitive erythropoiesis has been difficult to study, in part
because of the relative inaccessibility of the mammalian embryo
and because EryP form at a time when the embryo is still extremely
small. In addition, in the human embryo, the time during embryonic development at which blood islands first emerge (days 16 and
17, Figure 1B) is too early to be accessible from elective
terminations of pregnancy.3 This difficulty underscores the value of
the ES cell system for investigations into these early embryonic
events.41 Past midgestation, maturing EryP and adult-type erythrocytes are present simultaneously within the circulation. The single
monoclonal antibody that specifically recognizes mouse erythroid
cells, Ter-119, binds to both EryP and EryD. To circumvent these
problems, we have used human ε-globin regulatory elements to
In the mouse, erythroid progenitors are found only in the yolk sac,
but not in the embryo proper, between E7.25 and E9.0.7,12 Primitive
erythropoiesis may involve a limited hierarchy of progenitors in
which a bipotential megakaryocyte-erythroid progenitor gives rise
to unipotential EryP progenitors. In the mouse embryo, the first
megakaryocytes, identified by staining for surface GP1b␤, are
found in the yolk sac at E9.5 and in the blood at E10.5, substantially
later than primitive erythroblasts.6 It is not clear whether the
apparent temporal difference in emergence of these 2 lineages
reflects a limitation in this method of detection of megakaryocytes
or a true kinetic difference in maturation of their progenitors. In any
case, EryP are the first morphologically identifiable hematopoietic
cells of the embryo.
The signals that initiate differentiation of EryP progenitors,
apparently just as the cells begin to enter the bloodstream,7,43 are
unknown. EryP progenitors were initially identified retrospectively
by their ability to form red-pigmented colonies in cultures containing semisolid medium supplemented with erythropoietin44 and
were termed EryP-CFC.7 The identification and isolation of these
progenitors have presented challenges because of a lack of specific
cell surface markers. Expression of CD31 (PECAM1), Tie-2,
CD34, and VE-cadherin45 or low level expression of CD4146 has
been used to enrich for EryP progenitors from E7.0 to E7.5
embryos, but these proteins are also expressed on endothelial
and/or definitive hematopoietic progenitors.
Recently, it has been possible to isolate EryP progenitors
prospectively from E7.5 to E8.5 embryos12 on the basis of an
EryP-specific GFP reporter expressed in mice.42 In this line,
expression of a nuclear histone H2B-GFP fusion protein is driven
by a human embryonic ε-globin promoter and sequences from the
␤-globin locus control region42 (Figure 5). The fusion protein coats
chromatin and remains detectable through all phases of the cell
cycle (Figure 5C). Thus, it permits tagging and tracking of EryP
nuclei and identification of mitotic cells and has proven to be a very
useful model for studying embryonic erythropoiesis.12,39,42,47 GFP
fluorescence (and endogenous expression of embryonic globin
genes) was detected in mouse embryos as early as E7.512; therefore,
Figure 4. Maturational globin gene switching in developing primitive erythroid cells. “Maturational” globin
gene switching40 refers to sequential changes in expression from ␤h1- to ␧Y- (both embryonic) to adult ␤maj- (␤1)
globin within the primitive erythroid lineage during its
differentiation. (A) Relative expression is shown for the
3 ␤-like globin genes. ␧Y-globin remains the predominant
transcript throughout EryP development; even at late
times, ␤maj-globin expression remains low. (B) The data
are presented in a normalized form, with peak expression
set at 100 for each gene, to emphasize their sequential
expression within maturing EryP from E8.5 to E12.5 (J.I.,
Z. He, M.H.B., unpublished data, February 2009).
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BLOOD, 24 MAY 2012 䡠 VOLUME 119, NUMBER 21
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4831
Figure 5. Nuclear GFP reporter for EryP. (A) Histone
H2B-GFP expression within the yolk sac “blood islands”
of an E8.5 ␧-globin-H2B-GFP transgenic embryo.
(B) GFP(⫹) cells within the yolk sac vasculature of an
E9.5 ␧-globin-H2B-GFP transgenic embryo. (C) Nuclear
expression of H2B-GFP fusion protein permits identification of mitotic figures and actively dividing cells. Blue
represents 4,6-diamidino-2-phenylindole. Embryos were
imaged as in Isern et al.12,39
transgene expression is activated toward the end of gastrulation, at
the time when the first erythroid cells are specified from nascent
mesoderm.28 At E7.5 and E8.5, a substantial proportion (⬃ 15%20% and 50%, respectively) of all cells in the embryo expressed
GFP, indicating that significant resources are set aside by the
developing embryo for this single lineage.12 EryP progenitor
activity, evaluated by quantifying EryP-CFC, was found exclusively in the FACS-sorted GFP-positive population and expanded
rapidly between E7.5 and E8.5.12 A subset of GFP-positive cells
from E7.5 embryos expressed Flk1, c-kit, VE-cadherin, and Tie-2
proteins, all normally associated with endothelial cells.12 All
progenitor activity was found in the fraction of GFP-positive cells
that expressed c-kit, whereas Tie-2 expression provided a 4-fold
enrichment of progenitors in the GFP-positive population.12 Although progenitor activity is lost once EryP begin to enter the
bloodstream,43 circulating EryP do continue to divide until around
E13.5 (N. Neriec, S.T.F., M.H.B., unpublished data, August
2006).37
Interestingly, a variety of cell adhesion proteins (Pecam1,
CD44, and the integrins ␣4, ␣5, ␤1, and ␤3) are found on the
surface of EryP progenitors.12 Genes encoding additional adhesion
molecules as well as collagens are also expressed in these cells.12
Therefore, within the yolk sac, EryP may form tight associations
with each other and/or with surrounding endothelial cells, as
suggested by electron microscopy.48 Loss of adhesion proteins
from the surface of EryP in the yolk sac, along with expression of
metalloproteases, might help to untether the cells and facilitate
their entry into the bloodstream.12
Transition from yolk sac to circulation stage
EryP
Shortly before midgestation in the mouse embryo, the circulatory
systems of the embryo, yolk sac, and placenta connect. As the heart
begins to beat (⬃ E9.0), proerythroblastic EryP enter the circulation as a synchronous cohort of nucleated cells.43 Circulating EryP
in rodents continue to mature within the bloodstream.13,14,38 They
display a developmental progression from proerythroblast to
orthochromatic erythroblast to reticulocyte that is characterized by
loss of nucleoli (E9.5-E10.5), decreased cell diameter and crosssectional area (E10.5-E11.5), condensation of nuclei to 20% of
their original size (E10.5 onwards), expression of cell adhesion
proteins (E12.5-E14.5),13,42 and enucleation to primitive reticulocytes (E12.5 onwards)13,14 (Figure 3). Thus, the maturation of
primitive erythroblasts is stepwise and developmentally synchronized. EryP maturation is reflected in a progressive loss of CD71
and up-regulation of Ter-119 expression,13 analogous to that
observed for fetal liver erythropoiesis.49 Proteins, such as ␤1
integrin and phosphatidylserine, are selectively partitioned onto the
membrane surrounding the expelled nuclei of adult erythroid
cells.50-52 In contrast, Ter-119 is partitioned to the reticulocyte
membrane.51 Selective reorganization of surface proteins, such as
Ter-119, CD71, and several integrins, occurs during enucleation of
EryP as well.13,42 We have speculated that loss of adhesion proteins
during maturation may facilitate entry of the reticulocyte into the
circulation and that preferential coating of the extruded nucleus
may facilitate its engulfment by macrophages.42 Figure 6 contains a
summary of these changes.
The possibility that EryP enucleate was suggested by 2 reports
from the 1970s describing enucleated megalocytic, hemoglobincontaining cells in the bloodstream.37 Four decades later, an
antibody against a mouse embryonic ␤-like hemoglobin chain was
used to demonstrate that EryP begin to undergo nuclear extrusion
by E12.5.14 These observations were confirmed using a flow
cytometric method in which nucleated and enucleated EryP could
be distinguished and quantified in blood on the basis of binding of
the cell-permeable DNA-binding fluorescent dye DRAQ5 and
EryP-specific expression of GFP.13 Analysis of the kinetics of
enucleation revealed a precipitous (50%) reduction in nucleated
EryP between E13.5 and E14.5; this process was nearly complete
by E15.5.13 However, the total number of GFP-positive EryP cells
remained approximately the same from E12.5 to the end of
gestation (R. Moore, S.T.F., M.H.B., unpublished data, October
Figure 6. Model for protein redistribution on surface
of maturing erythroid cells. As EryP prepare to enucleate, redistribution of surface antigens (Ter119, orange; ␣4
and ␤1 integrins, blue) occurs such that the reticulocyte
(enucleated erythroid cell) is decorated with Ter119 but
displays little, if any, ␣4 and ␤1 integrin. Conversely, the
extruded nucleus is preferentially coated with ␣4 and ␤1
integrins, perhaps facilitating engulfment by macrophages.42 Protein redistribution has been described for
maturing adult erythroblasts as well.51
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BARON et al
BLOOD, 24 MAY 2012 䡠 VOLUME 119, NUMBER 21
Figure 7. Stages of primitive erythropoiesis. Summary of different phases of EryP development, from
progenitor to bloodstream (where the cells continue to
undergo limited proliferation) to terminal maturation and
enucleation. The images of EryP at different stages were
cropped from photographs of actual Giemsa-stained
cells.
2009).13 Thus, in contrast with the prevailing view that EryP
survive for only 4 or 5 days and gradually disappear during
gestation, perhaps by apoptosis,53,54 these experiments indicated
that EryP are a stable population and that enucleation does not
foreshadow their death.13 For at least 3 weeks after birth, approximately 0.5% of all cells in the bloodstream are GFP-positive
EryP.13 It seems likely that the life span of a primitive erythrocyte is
similar to that of an adult red blood cell and that EryP, like their
adult counterparts, are eventually cleared by the spleen.
Niches for primitive erythroid maturation and
enucleation
In contrast with definitive erythrocytes, which are released directly
into the circulation from the fetal liver or adult bone marrow as
enucleated cells, EryP enter the blood with their nuclei intact, at
around E9.0, with the initiation of a strong heartbeat.43 EryP
eventually extrude their nuclei, but, surprisingly, the first enucleated EryP are not detectable in the blood for at least 3 days (E12.5)
after the cells have begun to circulate.13,14 This phase of primitive
erythropoiesis is nearly complete by approximately E15.5.13,14 An
explanation for this lag may be that EryP appear to collect and
complete their maturation within the fetal liver,42,55 an organ that
does not form until midgestation.
Detailed analysis of the surface phenotype of GFP-positive
EryP revealed that integrins and other cell surface adhesion
proteins are expressed on EryP beginning around E12.5, reflecting
a previously unrecognized aspect of their maturation13 and suggesting that they may home to a fetal tissue, such as the liver, where
they would continue to mature. Transgenic mouse lines expressing
pancellular13 or nuclear42 (Figure 5) GFP were used to test this
hypothesis. The fetal liver is the site of development of adult-type
red blood cells, which mature within “erythroblastic islands”
(EBIs).56 EBIs, which are also found in bone marrow and spleen,
are 3-dimensional structures containing a central macrophage
surrounded by erythroid cells at various stages of maturation.56
Cytoplasmic extensions from the macrophage make intimate
contact with the erythroblasts, leading to the suggestion that the
macrophages function as nurse cells and also engulf expelled
erythroid nuclei.56
EryP appear to collect in the fetal liver and to integrate into
EBIs, along with developing definitive erythroblasts.42 The EryP
found in the fetal liver had uniformly and strongly up-regulated a
range of adhesion molecules, including ␣4, ␣5, and ␤1 integrins
and CD44, and showed greater adhesion to macrophages in
reconstituted EBIs than did circulating EryP.42 The “rosettes”
formed by these interactions were disrupted by a blocking antibody
against VCAM-1, the macrophage receptor for ␣4␤1 integrin42 and
by peptide blocking of fibronectin binding by ␣4 and ␣5 integrins
(J.I. and M.H.B., unpublished data, April 2008). Disruption of
EryP-macrophage interactions using ␣4 integrin were reported by
one group55 but was not observed by us, for blocking antibodies
against either ␣4 or ␤1 integrin.42 Confocal microscopy and FACS
analyses of fetal livers from the ε-globin–H2B-GFP (nuclear GFP)
transgenic mice indicated that nuclei extruded by EryP, like those
of adult red blood cells,52 undergo phagocytosis by macrophages.42
In both adult and embryonic lineages, nuclear extrusion occurs by
budding off of the highly condensed nucleus surrounded by a rim of
plasma membrane.55
The nuclear-GFP reporter made it possible to monitor EryP
enucleation at high resolution using flow cytometry. Bright GFPpositive structures with very low granularity were FACS-purified
from the bloodstream and fetal liver and displayed the morphology
expected for expelled nuclei, with a surrounding remnant of
cytoplasm and a cell membrane.42 Using antibodies against mouse
embryonic ␤-hemoglobins to identify EryP, others have also found
that these cells can interact with macrophages, which can engulf
the expelled nuclei in vitro.55 The GFP fluorescent signal allowed
us to sort and analyze the extruded nuclei, which were highly
enriched for integrins, in contrast with the enucleated primitive
reticulocytes that showed little or no expression of these proteins.42
Therefore, adhesion molecules are specifically redistributed onto
the membrane surrounding the expelled nucleus, perhaps causing it
to become more attractive for engulfment by fetal liver macrophages42 (Figure 6). Protein sorting has also been described for
enucleating adult erythoblasts.51 The stages of primitive erythropoietic development are summarized in Figure 7.
Although the numbers of free GFP-positive EryP nuclei detected in the circulation were lower than in the fetal liver,
suggesting that a small fraction briefly escape engulfment by
macrophages and enter the bloodstream, it is also possible that
some EryP enucleate while in circulation. The free nuclei presumably are phagocytosed later, when they circulate through the fetal
liver or other tissues,42 possibly including the placenta, as reported
for the human embryo.57 Human primitive erythroblasts also
enucleate, but the site is apparently the placental villi rather than
the fetal liver.57 Microscopic analysis suggests that the nuclei
expelled by human EryP are engulfed by macrophages, although
this finding remains to be confirmed more directly57 (eg, using
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FACS). Interestingly, macrophage progenitors are present in the
human placenta before the onset of circulation.57 Whether enucleation of EryP also occurs in the mouse placenta is not known.
Collectively, these results indicate that one of the major roles of
fetal liver macrophages is the engulfment and destruction of
erythroid nuclei. Although a role for macrophages in definitive
erythroid cell maturation is well accepted,56 several lines of
evidence suggest that macrophages are not essential for their
enucleation in vivo.58 Enhancement of EryP enucleation during
coculture on fetal liver macrophages was detected in one study55
but not in another.42 Nevertheless, interactions with macrophages
may facilitate other aspects of erythroid maturation. For example,
erythroid progenitors expand and mature more efficiently in vitro
in the presence of stromal cells,59 and definitive erythroblast
proliferation is stimulated by coculture with macrophages.60
the developing fetal liver, thymus, and bone marrow, presumably as
niches form and acquire the ability to support HSC selfrenewal.1,2,9 The final wave of definitive hematopoiesis produces
all lineages, including B and T lymphoid potentials.1,2
Development of the hematopoietic system in humans is less
well studied but is broadly similar to that of the mouse.3,9 In the
human embryo, erythroid burst-forming unit activity is first
detected at 7 to 8 weeks, after the second wave of colonization of
the fetal liver by HSCs3 (Figure 1B). Fetal and adult human
erythrocytes can be distinguished by their production of distinct
forms of hemoglobin (hemoglobin F or A, respectively).34 The
switch in expression of fetal to adult ␤-like globin genes begins at
approximately 32 weeks and is completed after birth.34 In the
human embryo, as in the mouse, HSCs have been identified in the
aorta-gonad-mesonephros region, large vessels, and placenta.65
Emergence of definitive erythroid lineages in
the yolk sac and fetal liver
Growth factor requirements for
erythropoiesis
The fetal liver provides a microenvironment(s) for the expansion
and differentiation of definitive HSCs, from which definitive
erythroid cells differentiate via a hierarchy of progenitors.1,2 For
many years, the first definitive erythroid cells were thought to
originate from HSCs that colonize the fetal liver.1,2,61 However, a
second wave of hematopoiesis was found to emerge from the yolk
sac at around E8.25 to E8.5; it produces cells of the definitive
erythroid lineage, identified in vitro using an assay for erythroid
burst-forming units, as well as megakaryocyte and myeloid lineages.6-8,62 The second wave of hematopoiesis also includes
multipotential, highly proliferative progenitors (HPP-CFC) identified in vitro.62 HPP-CFC and more extensively self-renewing
erythroid progenitors that expand in culture63 are both found in the
yolk sac at about the same time of development. These progenitors
are maintained in culture under different growth factor and
cytokine conditions,62,63 however, and their lineage relationship
in vivo (if any) is unclear. In addition, HPP-CFC lack HSC activity
and are found in the yolk sac 12 to 24 hours earlier than the first
newborn repopulating HSCs64 are detected (M. Yoder, personal
oral communication, October 2011).
Recently, it has been demonstrated that erythropoiesis in the
fetal liver begins before its colonization by HSCs.36 Early definitive
erythroblasts were isolated by flow cytometry on the basis of
expression of c-kit (which is not found on EryP at this stage of their
development12,13) and shown to display a distinct and unique
profile of endogenous mouse and transgenic human globin gene
expression.36 Maturing definitive erythroblasts isolated from E9.5
yolk sac show the same pattern of ␤-globin gene expression as the
early (E11.5) definitive fetal liver erythroblasts. This observation
and several other lines of circumstantial evidence suggest that the
first definitive erythroblasts in the fetal liver arise from yolk sac
erythroid/myeloid progenitors,36 although this remains to be demonstrated directly. It has been proposed that erythroid/myeloid
progenitor-derived definitive erythropoiesis bridges the transition
between primitive and HSC-derived erythropoiesis.2,36 It is tempting to speculate that the first hepatic colonization described for the
human fetus (Figure 1B) might correspond to the second wave of
hematopoiesis identified in the mouse embryo.
Definitive HSCs arise in the mouse embryo in the para-aortic
splanchnopleure (E7.5-E9.5), the aorta-gonad-mesonephros region
(E10.5-E11.5), the major blood vessels, and in the placenta.1,2,9 The
HSCs that form in these tissues do not differentiate there but seed
Little is known about the growth factor requirements for primitive
erythropoiesis in vivo. The hormone erythropoietin plays an
essential role in definitive erythropoiesis, providing a key survival
signal for late progenitors (colony-forming units-E).66 Mouse
embryos deficient for the erythropoietin receptor have measurable
but somewhat late defects in the proliferation and maturation of
primitive erythroblasts but not progenitors, becoming evident only
around E10.5 and later.67 Embryonic death from severe anemia
occurs by E13.5, with a nearly complete ablation of definitive
erythropoiesis.67,68 The differential effects of loss of erythropoietin
receptor signaling in the 2 lineages suggests a role for additional
cytokines in EryP development.37
Stem cell factor (or kit ligand) also has a differential effect on
EryP and EryD in mouse. Stem cell factor receptor (c-kit) mutants
die of severe anemia because of a failure in definitive erythropoiesis.69 Although c-kit is expressed on the surface of EryP progenitors,12,45 their proliferation, survival, and maturation are unaffected
by exogenous stem cell factor in culture.12 EryP progenitors
express another receptor tyrosine kinase receptor, Tie-2,12,45 but
their growth was not consistently stimulated by addition of a
recombinant Tie2 ligand, angiopoietin-1.12 It is possible that
functions for c-kit and/or Tie-2 signaling could be unmasked by
development of serum-free culture conditions for EryP progenitors.12
The TGF-␤ pathway has been implicated in embryonic erythroid development. On certain genetic backgrounds, embryos
deficient for TGF-␤1 die by midgestation from hematopoietic and
vascular defects70; this phenotype is regulated by a genetic
modifier.71 It is not known whether the hematopoietic defects
observed70 reflect abnormalities in the formation and/or proliferation of EryP progenitors or their maturation. Targeted mutation of
the Tgfbr1 receptor gene also results in embryonic lethality, but in
this case it was concluded that the defect involved angiogenesis and
that hematopoiesis was normal.72 However, the mutant embryos
were clearly anemic and EryP progenitor assays were not performed.72 Deletion of Tgfbr2 also resulted in hematopoietic and
vascular defects in the yolk sac.73 These embryos clearly had
greatly reduced numbers of EryP in the vessels of sectioned tissue,
but no further analysis was performed. Findings from our geneexpression profiling study prompted us to reevaluate the possibility
of a role for TGF-␤ signaling in the primitive erythroid lineage.
EryP progenitors express mRNAs for receptors (Tgfbr1 and
Tgfbr3, Acvr2b) and downstream signaling components (eg, Smad3,
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4834
BARON et al
Smad4, and Smad5) of these pathways; transcription of these genes
begins to decrease by E8.5.12 To determine whether the TGF-␤
signaling pathway can modulate the expansion of EryP progenitors,
we tested recombinant forms of the 3 Tgfbr1 ligands TGF-␤1,
TGF-␤2, and TGF-␤3 in clonogenic assays. TGF-␤1 (but not
TGF-␤2 or TGF-␤3) showed a dose-dependent effect on EryP
progenitor activity in vitro: growth of EryP progenitors was
inhibited at a low concentration and stimulated at higher concentrations of this cytokine.12 The TGF-␤ signaling pathway may
function via an autocrine or paracrine mechanism, as both the
receptor and its ligand, Tgf␤1, are transcribed in EryP.12 In contrast,
TGF-␤1 has been viewed as an inhibitor of definitive erythropoiesis in vitro, decreasing proliferation of progenitors and inducing
their differentiation.74 A more detailed evaluation of the function of
this pathway in EryP is warranted.
Fibroblast growth factor receptor-1 is required for hematopoietic development in differentiating mouse ES cells75 but whether
the absence of the receptor affects primitive and/or definitive
erythroid lineages was not defined. Sprouty (Spry) proteins antagonize signaling by receptor tyrosine kinases, such as fibroblast
growth factors and epidermal growth factor.76 A recent study
concluded that Spry1 negatively regulates primitive erythropoiesis
in mouse embryos, but EryP progenitor assays were not performed77 and the cellular basis for the defect needs to be clarified.
Metabolic changes during EryP development
EryP must adapt to changes in oxygen and nutrient supplies during
their maturation within successive microenvironments (yolk sac,
circulation, and fetal liver) within the embryo.42 The expression
profile of EryP is consistent with high glycolytic activity in
progenitors, followed by a metabolic switch as a group of genes
known to play crucial roles in glucose metabolism are rapidly
down-regulated around the time when these cells begin to circulate.12 In culture, not only are EryP progenitor numbers increased
under conditions of low oxygen but the colonies that form in
methylcellulose are also larger, apparently because of increased
EryP-CFC proliferation.12 EryP progenitors also display a signature
characteristic of the “Warburg effect” associated with rapidly
proliferating cells (eg, cancer cells) that catabolize glucose aerobically and produce high levels of lactate in the cytosol, rather than
using the more energy efficient mitochondrial pathway.78,79 We
propose that EryP switch from aerobic glycolysis to mitochondrial
oxidation as they enter the circulation. The rapid proliferation of
EryP progenitors and their cellular response to hypoxia are
reminiscent of stress erythropoiesis in the fetus and adult.80,81 It is
not known whether the aerobic glycolytic profile of EryP progenitors simply reflects the particular energy demands of these rapidly
dividing cells or is a more unique feature of primitive
erythropoiesis.12
Transcriptional regulation of erythropoiesis
The specification and differentiation of erythroid cells have been
well studied for the definitive lineage and are regulated at
transcriptional, posttranscriptional, and epigenetic levels. This
subject has been extensively reviewed by others.1,82-85 In this
section, we emphasize key points and insights from the recent
literature. The zinc finger protein GATA1 was one of the first cell
BLOOD, 24 MAY 2012 䡠 VOLUME 119, NUMBER 21
type-restricted transcription factors to be identified and cloned for
any lineage.1 Since that time, a battery of additional erythroid
regulators have been discovered; the reader is referred to excellent
“primers” on core erythroid transcription factors (notably, GATA1,
EKLF/KLF1, NFE2, and SCL) and cofactors (FOG1, CBP/p300,
TRAP200, BRG1, and others).82-84
Genome-wide expression profiling revealed that transcription in
maturing EryP is characterized by 2 discrete waves that correlate
with key developmental hallmarks.12 GATA1 and EKLF are central
players in both primitive and definitive erythropoiesis. GATA1 and
GATA2 overlap in function in primitive erythropoiesis, as demonstrated by analysis of double-knockout embryos.86 EKLF was
initially thought to function primarily, if not exclusively, in
definitive erythropoiesis,87,88 although its expression had been
detected in mouse embryos as early as E7.5.89 It was later
appreciated that Eklf functions in primitive erythropoiesis as
well.53,90 We were able to analyze the impact of loss of one or both
alleles of Eklf during primitive erythropoiesis by crossing the
ε-globin:H2B-GFP transgenic mouse42 onto an Eklf mutant background.88 Haploinsufficiency of Eklf had a profound effect on the
surface phenotype of EryP but not EryD, suggesting that the
differentiation of EryP may be more sensitive to gene dosage than
that of EryD.39 Indeed, the levels of Eklf are approximately 3-fold
lower in EryP than in EryD.91 However, fetal-to-adult globin gene
switching is also sensitive to Eklf gene dosage91,92: haploinsufficiency of EKLF in humans causes hereditary persistence of fetal
hemoglobin.92
EryP and EryD differ in their requirements for a variety of other
transcriptional regulators. For example, in contrast with fetal liver
erythropoiesis, which is dependent on the function of the protooncogene c-Myb, the core binding factor Ruxn1, and the Kruppeltype zinc finger protein ZBP-89, primitive erythropoiesis is not.93-96
However, although it is not crucial for primitive erythropoiesis,
Runx1 does function in the development of EryP.97 The ε-globin:
H2B-GFP transgenic mouse42 should be useful in identifying
functions for additional transcription factors that may not be
essential in EryP. Conversely, lineage-specific ablation of genes in
EryP will permit the identification of genes that are critical for
primitive but not definitive erythropoiesis.
Both the primitive and definitive erythroid and megakaryocyte
lineages share a common progenitor (megakaryocyte-erythroid
progenitor).98 Eklf had been shown to play a dual role in definitive
hematopoiesis by promoting erythropoiesis and suppressing megakaryopoiesis.98,99 We found that null mutation of Eklf resulted in a
partial “identity crisis”39 within maturing EryP in the circulation,
with up-regulation of a subset of megakaryocyte-related surface
proteins, such as CD41 and Pecam-1/CD31, and transcription
factor genes, such as Fli-1.39 Therefore, loss of Eklf in EryP causes
a partial change in cellular identity.39 Additional observations12
suggest that Eklf not only regulates lineage divergence from the
megakaryocyte-erythroid progenitor but also controls various aspects of EryP maturation.12 Findings such as these underscore the
utility of the ε-globin–H2B-GFP transgenic mouse line42 for
evaluating the effects of targeted mutations specifically in the
primitive erythroid lineage.
It is now appreciated that not only the absolute and relative
levels of these proteins but also their order of action is critical in the
segregation of erythroid and megakaryocytic lineages as well as in
their differentiation.1,84,100,101 For example, conditional deletion of
Gata1 and Zfpm1 (which encodes FOG1) revealed that FOG1 and
GATA1 function sequentially in the formation and lineage divergence of erythroid-megakaryocytic progenitors during definitive
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BLOOD, 24 MAY 2012 䡠 VOLUME 119, NUMBER 21
MAMMALIAN EMBRYONIC ERYTHROPOIESIS
hematopoiesis.101 GATA1 and EKLF form large complexes with
many other proteins to regulate the expression of erythroid-specific
genes.102,103
ChIP-Seq, in which chromatin immunoprecipitation is coupled
with high-throughput DNA sequencing, has made it possible to
generate genome-wide maps of binding by transcription factors,
such as GATA1, GATA2, and NF-E2,104,105 and thereby define the
full repertoire of loci occupied by these factors. Advances in this
technology have allowed the identification of much wider combinatorial interactions among transcription factors than was previously
possible.102,103 Although detailed analysis of individual transcription factors has provided a wealth of information about their
functions in erythroid cells, there has been increasing emphasis on
their coordinated binding. It would be of interest to compare the
transcription complexes that form at different stages of EryP
development (Figure 4) with those found in differentiating EryD.
In conclusion, although erythropoiesis has traditionally been
described as encompassing embryonic (primitive) and “adult-type”
(definitive) phases, it now appears that there are not 2, but 3 distinct
erythroid lineages, each with a unique pattern of ␤-globin gene
expression.36 Thus, the erythroid burst-forming units that arise
from HSCs in the fetal liver actually constitute a third wave of
erythropoiesis. In addition, at least in the mouse, switching in
globin gene expression during development reflects maturational
switches within a single erythroid lineage36,40 (Figure 4) as well as
lineage switching (Figure 1). The progenitors that produce EryP are
the first hematopoietic cells of the embryo and are found in the yolk
sac only briefly but terminally differentiated primitive erythrocytes
are present in the circulation through the end of gestation and
probably for a short time after birth13 (primitive erythropoiesis is
summarized in Figure 7). The ontogeny of erythropoiesis is much
more complex than previously appreciated. It will be important to
take this complexity into consideration when interpreting observations made not only in the embryo/fetus but in cell culture systems,
such as ES and iPS cells. Comparative studies of erythropoiesis at
different stages of development, in different tissues within the same
stage, and in different species will probably continue to lead to
refinements in our understanding of the biology of the red blood
cell lineage.
Some of the pathways involved in the maturation of EryP and
EryD are probably conserved. Surprisingly little is known about
4835
how red blood cells enucleate, even in the adult. Similarly, the
mechanisms underlying the redistribution of proteins on the surface
of erythroid cells as they mature (Figure 6) have barely been
explored. Comparisons between primitive and definitive erythropoiesis may provide useful insights into these processes, our understanding of which remains largely descriptive. Elucidation of the
common as well as the distinguishing features of embryonic versus
adult erythroid development will be a prerequisite for the directed
differentiation of human stem or progenitor cells for therapeutic
purposes and for the efficient production of pure populations of red
blood cells for transfusion in patients. The study of red blood cells
will probably continue to provide fascinating insights into fundamental questions in biology and information of considerable
practical value.
Acknowledgments
This work was supported in part by the National Institutes of
Health (grants RO1 HL62248, DK52191, and EB02209), the
Roche Foundation for Anemia Research (grant 9699367999, cycle
X), and the New York State Department of Health (NYSTEM grant
N08G-024, M.H.B.).
Authorship
Contribution: M.H.B. created figures and wrote the paper; J.I.
created figures; and S.T.F. wrote an initial draft of the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
The current affiliation for J.I. is Cardiovascular Development
and Repair Department, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain. The current affiliation for S.T.F. is
Disciplines of Physiology, Anatomy and Histology, School of
Medical Sciences, University of Sydney, Camperdown NSW,
Australia.
Correspondence: Margaret H. Baron, Mount Sinai School of
Medicine, Box 1079, 1468 Madison Ave, Annenberg 24-04E, New
York, NY 10029-6574; e-mail: [email protected].
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2012 119: 4828-4837
doi:10.1182/blood-2012-01-153486 originally published
online February 15, 2012
The embryonic origins of erythropoiesis in mammals
Margaret H. Baron, Joan Isern and Stuart T. Fraser
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