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Brief report
Polarized expression of bone morphogenetic protein-4 in the human
aorta-gonad-mesonephros region
Caroline J. Marshall, Christine Kinnon, and Adrian J. Thrasher
In the mammal, definitive hematopoietic
stem cells (HSCs) are first derived from
mesodermal cells within a region of the
embryonic para-aortic splanchnopleura
known as the aorta-gonad-mesonephros
(AGM). Within this region, HSCs are
thought to arise from hemangioblast precursors located in the ventral wall of the
dorsal aorta. However, the factors that
regulate HSC development in vivo are still
largely unknown. Bone morphogenetic
protein (BMP)-4, a member of the trans-
forming growth factor beta (TGF-␤) superfamily of growth factors, is a potent ventralizing factor and has been implicated in
the commitment of embryonic mesodermal cells to a hematopoietic fate in a
number of systems. In the human AGM,
we find that BMP-4 is expressed at high
levels, and with striking polarity, in a
region of densely packed cells underlying
intra-aortic hematopoietic clusters. In contrast, TGF-␤1 is expressed predominantly
by hematopoietic cells within the clus-
ters. These findings implicate both BMP-4
and TGF-␤1 in the initiation and regulation of hematopoiesis in the human AGM.
Furthermore, the distribution of BMP-4
expression is highly suggestive of a direct role in the specification of human hematopoietic cells from embryonic mesoderm in
vivo. (Blood. 2000;96:1591-1593)
© 2000 by The American Society of Hematology
Introduction
The major source of definitive murine HSCs has been mapped to
the embryonic AGM, at a time when clusters of hematopoietic cells
are found adhering to the ventral wall of the dorsal aorta.1,2 The
appearance of clusters is restricted to between 9.5 to 11.5 days post
coitum in the mouse and 30 to 37 days gestation in the human.3,4
These cell clusters express a number of molecules in common with
endothelial cells lining the wall of the dorsal aorta, including the
membrane glycoprotein CD34, the transcription factors SCL and
GATA-2, and the growth factors receptor c-Kit and FLK-1.5,6 The
shared expression patterns and the close physical association
between the HSC cluster and the endothelium has led to the
suggestion that both lineages arise from a common hemangioblast
precursor located within or underlying the wall of the dorsal aorta.
In addition, the marked asymmetry of this process suggests that
cluster formation is not a stochastic event, but one that is
determined by microenvironmental signals localized to the ventral
aortic wall.
Thus far, the factors that regulate hemangioblast differentiation
in vivo remain unknown although analysis of hematopoietic
development in lower vertebrates suggests that members of the
TGF-␤ superfamily of secreted polypeptide growth factors, including the bone morphogenetic proteins (BMP) play a critical role.
During embryogenesis, BMPs are thought to specify a variety of
cell fates dependent on concentration gradients, and expression of
antagonistic factors such as activin and noggin.7 One family
member, BMP-4, is known to induce ventral mesoderm, the tissue
that will eventually give rise to the hematopoietic system. Evidence
from studies using Xenopus laevis suggests that BMP-4 is also
involved in the commitment of mesodermal cells to the blood
lineage.8 In Xenopus, HSCs arise in the embryonic ventral meso-
derm, which contains the dorsal lateral plate, a region analogous to
the mammalian AGM. Ectoderm-derived animal cap cells, which
do not normally give rise to blood cells, can be induced to do so in
vivo after treatment with ectopic BMP-4. After mesoderm has
formed, expression of a dominant negative BMP-4 receptor inhibits
normal hematopoietic development, and mutant embryos lacking
BMP-4 expression also fail to develop hematopoietic tissue. These
data support a role for BMP-4 not only in mesoderm formation but
also in the conversion of mesoderm to blood, possibly by regulating the temporal and spatial expression of necessary transcription
factors within the hemangioblast population.
The involvement of BMP-4 in the initiation of mammalian
hematopoiesis has been less easy to study. For example, mice
lacking BMP-4 seldom develop beyond the egg cylinder stage,
preceding the appearance of blood cells because of a critical
requirement during gastrulation. However, in rare cases where
homozygous mutants have developed to later stages, there is a
noticeable paucity of blood cells circulating in the vasculature.10
On the other hand, the addition of BMP-4 to murine embryonic
stem (ES) cells results in the acquisition of mesodermal markers
and development of myeloid hematopoietic precursors. Interestingly, the dose of BMP-4 required for mesoderm formation from
ES cells is considerably lower than that required for maximal
hematopoietic output, suggesting that BMP-4 is sufficient to
initiate hematopoiesis from existing mesodermal tissues, but in a
concentration dependent manner.11
Unlike BMPs, TGF-␤1 itself is not thought to be directly
associated with dorsoventral patterning, but is an important regulator of cell proliferation and differentiation in a variety of tissues
during embryogenesis. Mice lacking TGF-␤1 mainly die in utero
From the Molecular Immunology Unit, Institute of Child Health, London, UK.
Health, 30 Guilford St, London, WC1N 1EH, United Kingdom; email:
[email protected].
Submitted February 7, 2000; accepted April 12, 2000.
Supported by Grants 044783/Z/95 and 057965/Z/99 from the Wellcome Trust,
UK. A.J.T. is a Wellcome Trust Senior Clinical Fellow.
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.
Reprints: Caroline J. Marshall, Molecular Immunology Unit, Institute of Child
© 2000 by The American Society of Hematology
BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
1591
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1592
MARSHALL et al
BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
between 9.5 to 11.5 days post coitum because of defects in yolk sac
hematopoiesis and vasculogenesis.12 In addition, TGF-␤1 has been
shown to inhibit the initial proliferation and differentiation of
murine long-term repopulating HSCs and primitive human longterm culture-initiating cells.13,14 Early human hematopoietic progenitors themselves secrete TGF-␤1, and it has been suggested that this
autocrine production may be important in the negative regulation
of cell cycling.15
Study design
Human tissue collection and processing
Human embryos and yolk sacs were obtained from the MRC-funded
Human Embryo Bank maintained at the Institute of Child Health. The
embryos were staged and processed as previously described.6
Immunohistochemistry
Paraffin-embedded tissue sections through human embryos at 28, 34, and
38 days gestation and human yolk sacs were pretreated and antibodybinding visualized as described previously.6 Transverse sections were
incubated with mouse monoclonal antibodies raised against human BMP-4
(clone 3H2: Novocastra Laboratories, UK) and TGF␤1 (clone TGFB17:
Novocastra Laboratories, UK). For TGF␤1, a high-temperature antigenunmasking technique was used.6 Sections were incubated with primary
antibody for 60 minutes at room temperature. Results shown are
representative.
Results and discussion
Immunohistochemical analysis of the human embryonic AGM
reveals clusters of hematopoietic cells associated with the ventral
wall of the dorsal aorta. These cells express the membrane
glycoprotein CD34 and the hematopoietic-specific marker CD45.4,6
We have also previously reported the presence of a morphologically distinct region resembling a stromal layer in the mesenchyme
directly beneath the ventral wall of the dorsal aorta in the AGM that
is associated with intra-aortic clusters. This consists of between 5
and 7 layers of densely packed cells in the 34-day human embryo
(Figure 1A and B) or 3 to 4 layers in the mouse at 10.5 days post
coitum, and expresses high levels of the extracellular matrix
protein tenascin-C (data not shown6). In view of their emerging
importance in the development of hematopoiesis, we investigated
whether expression of members of the TGF-␤ family is localized to
this region. In the 34-day human embryo, BMP-4 was expressed in
a gradient across the dorsoventral axis with many highly positive
cells distributed throughout the ventral mesoderm, including
transected descending neuronal tracts (Figure 1A). Within the
AGM region, compared with the surrounding tissues and adjacent
cardinal vessels, expression of BMP-4 was polarized to the ventral
wall of the dorsal aorta, specifically in the region of the “stromal”
layer and underlying the intra-aortic clusters (Figure 1B). At 28
days gestation, no distinct dorsoventral gradient of expression was
apparent, neuronal tracts were absent and BMP-4 expression within
the ventral mesoderm was generally low, with few strongly positive
cells (Figure 1C). However, at this earlier developmental stage,
emerging clusters of hematopoietic cells were also found adhering
to the ventral wall of the dorsal aorta and BMP-4 expression was
strikingly restricted to the “stromal” layer, which consisted of
only 2 or 3 layers of cells at this age, underlying these clusters
(Figure 1D).
Figure 1. Expression of transforming growth factors BMP-4 and TGF␤1 in the
human embryonic AGM region. Immunohistochemical analysis on transverse
sections of human embryos and yolk sac. (A) Within the AGM region at 34 days, a
gradient of BMP-4 protein exists across the dorsoventral axis at the level of the AGM
region. BMP-4 is expressed in the ventral mesoderm (below thin arrows), which
includes the dorsal aorta (da), cardinal veins (cv), and mesonephros (m), whereas
the dorsal mesoderm (above thin arrows), between the dorsal aorta and neural tube
(nt), is predominantly negative. (B) Higher magnification of (A) in the region of the
dorsal aorta. BMP-4 expression is polarized to the ventral stromal region (s)
underlying the intra-aortic hematopoietic cluster (hc). (C) In contrast, in a younger
28-day-old embryo, BMP-4 is expressed at low level in both ventral and dorsal
mesoderm, with few highly positive cells. (D) However, as shown by a higher
magnification of (C), BMP-4 expression is at this stage restricted to the thin “stromal”
layer (s) underlying the ventral wall of the aorta from which a cluster of cells (hc) can
be seen emerging. (E) At more caudal levels (34 days), no intra-aortic clusters are
visible and BMP-4 expression is markedly lower than in the AGM, consistent with an
anterior-posterior progression of development during embryogenesis. (F) At 38 days
gestation, BMP-4 expression within the AGM region is relatively uniform around the
entire aorta and is not polarized to the ventral wall. (G) Cells expressing BMP-4
(arrows) are specifically associated with blood islands (bi) in the embryonic yolk sac
(31 days). (H) TGF␤1 is expressed by cells within the intra-aortic cluster (hc).
Uniformly low level expression is detected around the aorta. (Original magnification:
A, C, ⫻15; B, D, E, G, H ⫻78, F ⫻36).
Outside the AGM, at more caudal and rostral locations, the level
of BMP-4 expression was comparatively low and no hematopoietic
clusters were observed (Figure 1E and data not shown). Similarly,
in older (38-day) embryos, after intra-aortic clusters have disappeared, BMP-4 expression around the dorsal aorta was no longer
polarized (Figure 1F). Interestingly, the analysis of human embryonic yolk sac also revealed strong BMP-4 expression in cells
specifically associated with blood islands (Figure 1G).
We also investigated the expression pattern of TGF-␤1. In
contrast to BMP-4, the expression of TGF-␤1 was uniformly low
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BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
POLARIZED BMP4 EXPRESSION IN THE HUMAN AGM
around the dorsal aorta, but detectable at higher levels in the
cytoplasm of hematopoietic cells within the intra-aortic clusters
(Figure 1H). No positive staining was observed in controls using
normal serum or secondary antibody only.
In summary, we find that BMP-4 is expressed at high levels in a
cell-dense region underlying the ventral wall of the dorsal aorta at
the time of human AGM hematopoiesis. Taken together with
evidence from other developmental systems, this may be a critical
requirement for the initiation of a hematopoietic differentiation
program from mesodermal precursors. The factors that regulate
expression of BMP-4, and downstream signaling events that
determine this process are at present unclear, but may involve
tightly regulated expression (spatial and temporal) of transcription
factors such as Tal-1/SCL, Lmo2, GATA-2, and AML1/Cbfa2, a
member of the family of core-binding transcription factors.16-20 For
example, in a parallel system, the induction of core-binding factor
AML3 by BMP family members has been shown to be tightly
linked to the process of osteoblastic differentiation.21,22 AML1 is
1593
now known to be required for definitive hematopoiesis23,24 and is
transiently expressed in cells in the ventral wall of the dorsal aorta
in the murine AGM coincident with the appearance of hematopoietic clusters.20 In the embryonic AGM, BMP-4 may therefore
directly influence the conversion of hemangioblasts to committed
HSCs through induction of AML1. The contrasting expression
patterns of BMP-4 and TGF-␤1 in the embryonic AGM are
consistent with a role for BMP-4 in the initiation of HSC
development from mesodermal precursors, and for TGF-␤1, possibly together with BMP-4, in the regulation of hematopoietic
cell fate.
Acknowledgment
We would like to thank Rachel Moore for preparation of embryonic
tissue used in this study.
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2000 96: 1591-1593
Polarized expression of bone morphogenetic protein-4 in the human
aorta-gonad-mesonephros region
Caroline J. Marshall, Christine Kinnon and Adrian J. Thrasher
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