Abstract

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 transforming growth factor beta (TGF-β) 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-β1 is expressed predominantly by hematopoietic cells within the clusters. These findings implicate both BMP-4 and TGF-β1 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.

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-β 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 laevissuggests that BMP-4 is also involved in the commitment of mesodermal cells to the blood lineage.8 InXenopus, HSCs arise in the embryonic ventral mesoderm, 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-β1 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-β1 mainly die in utero between 9.5 to 11.5 days post coitum because of defects in yolk sac hematopoiesis and vasculogenesis.12 In addition, TGF-β1 has been shown to inhibit the initial proliferation and differentiation of murine long-term repopulating HSCs and primitive human long-term culture-initiating cells.13,14 Early human hematopoietic progenitors themselves secrete TGF-β1, 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 antibody-binding visualized as described previously.6Transverse sections were incubated with mouse monoclonal antibodies raised against human BMP-4 (clone 3H2: Novocastra Laboratories, UK) and TGFβ1 (clone TGFB17: Novocastra Laboratories, UK). For TGFβ1, a high-temperature antigen-unmasking technique was used.6Sections 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 (Figure1A 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-β 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).

Fig. 1.

Expression of transforming growth factors BMP-4 and TGFβ1 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) TGFβ1 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).

Fig. 1.

Expression of transforming growth factors BMP-4 and TGFβ1 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) TGFβ1 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-β1. In contrast to BMP-4, the expression of TGF-β1 was uniformly low 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 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-β1 in the embryonic AGM are consistent with a role for BMP-4 in the initiation of HSC development from mesodermal precursors, and for TGF-β1, 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.

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.

References

References
1
Muller
AM
Medvinsky
A
Strouboulis
J
Grosveld
F
Dzierzak
E
Development of hematopoietic stem cell activity in the mouse embryo.
Immunity.
1
1994
291
301
2
Medvinsky
A
Dzierzak
E
Definitive hematopoiesis is autonomously initiated by the AGM region.
Cell.
86
1996
897
906
3
Wood
HB
May
G
Healy
L
Enver
T
Morriss-Kay
GM
CD34 expression patterns during early mouse development are related to modes of blood vessel formation and reveal additional sites of hematopoiesis.
Blood.
90
1997
2300
2311
4
Tavian
M
Coulombel
L
Luton
D
San Clemente
H
Dieterlen-Lievre
F
Peault
B
Aorta-associated CD34+ hematopoietic cells in the early human embryo.
Blood.
87
1996
67
72
5
Labastie
MC
Cortes
F
Romeo
PH
Dulac
C
Peault
B
Molecular identity of hematopoietic precursor cells emerging in the human embryo.
Blood.
92
1998
3624
3635
6
Marshall
CJ
Moore
RL
Thorogood
P
Brickell
PM
Kinnon
C
Thrasher
AJ
Detailed characterization of the human aorta-gonad-mesonephros region reveals morphological polarity resembling a hematopoietic stromal layer.
Dev Dyn.
215
1999
139
147
7
Hogan
BLM
Bone morphogenetic proteins: multifunctional regulators of vertebrate development.
Genes Dev.
10
1996
1580
1594
8
Huber
TL
Zhou
Y
Mead
PE
Zon
LI
Cooperative effects of growth factors involved in the induction of hematopoietic mesoderm.
Blood.
92
1998
4128
4137
9
Zhang
C
Evans
T
BMP-like signals are required after the midblastula transition for blood cell development.
Dev Genet.
18
1996
267
278
10
Winnier
G
Blessing
M
Labosky
PA
Hogan
BLM
Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse.
Genes Dev.
9
1995
2105
2116
11
Johansson
BM
Wiles
MV
Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development.
Mol Cell Biol.
15
1995
141
151
12
Dickson
MC
Martin
JS
Cousins
FM
Kulkarni
AB
Karlsson
S
Akhurst
RJ
Defective hematopoiesis and vasculogenesis in transforming growth factor β1 knock out mice.
Development.
121
1995
1845
1854
13
Sitnicka
E
Ruscetti
FW
Priestley
GV
Wolf
NS
Bartelmez
SH
Transforming growth factor β1 directly and reversibly inhibits the initial cell divisions of long-term repopulating hematopoietic stem cells.
Blood.
88
1996
82
88
14
Garbe
A
Spyridonidis
A
Mobest
D
Schmoor
C
Mertelsmann
R
Henschler
R
Transforming growth factor beta-1 delays formation of granulocyte-macrophage colony forming cells, but spares more primitive progenitors during ex vivo expansion of CD34+ haematopoietic progenitor cells.
Br J Haematol.
99
1997
951
958
15
Hatzfeld
J
Li
ML
Brown
EL
et al. 
Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor beta 1 or Rb oligonucleotides.
J Exp Med.
174
1991
925
929
16
Mead
PE
Kelley
CM
Hahn
PS
Piedad
O
Zon
LI
SCL specifies hematopoietic mesoderm in Xenopus embryos.
Development.
125
1998
2611
2620
17
Manaia
A
Lemarchandel
V
Klaine
M
et al. 
Lmo2 and GATA-3 associated expression in intraembryonic hemogenic sites.
Development.
127
2000
643
653
18
Maeno
M
Mead
PE
Kelley
C
et al. 
The role of BMP-4 and GATA-2 in the induction and differentaition of hematopoietic mesoderm in Xenopus leavis.
Blood.
88
1996
1965
1972
19
Minegishi
N
Ohta
J
Yamagiwa
H
et al. 
The mouse GATA-2 gene is expressed in the para-aortic splanchnopleura and aorta-gonads and mesonephros region.
Blood.
93
1999
4196
4207
20
North
T
Gu
TL
Stacy
T
et al. 
Cbfa2 is required for the formation of intra-aortic hematopoietic clusters.
Development.
126
1999
2563
2575
21
Tsuji
K
Ito
Y
Noda
M
Expression of PEBP2alphaA/AML3/CBFA1 gene is regulated by BMP4/7 heterodimer and its overexpression suppresses type 1 collagen and osteocalcin gene expression in osteoblastic and nonosteoblastic mesenchymal cells.
Bone.
22
1998
87
92
22
Lee
MH
Javed
A
Kim
HJ
et al. 
Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor beta 1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation.
J Cell Biochem.
73
1999
114
125
23
Okuda
T
van Duersen
J
Hiebert
SW
Grosveld
G
Downing
JR
AML1, the target of multiple chromosome translocations in human leukemia, is essential for normal fetal liver hematopoiesis.
Cell.
84
1996
321
320
24
Wang
Q
Stacy
T
Binder
M
Marin-Padilla
M
Sharpe
AH
Speck
NA
Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis.
Proc Natl Acad Sci U S A.
93
1996
3444
3449

Author notes

Caroline J. Marshall, Molecular Immunology Unit, Institute of Child Health, 30 Guilford St, London, WC1N 1EH, United Kingdom; email:c.marshall@ich.ucl.ac.uk.