1 Laboratory of Hematopoiesis and Leukemia, Clinical Research Institute of
Montreal, Montreal, Canada
2 Department of Medicine, Division of Experimental Medicine, McGill University,
Montreal, Canada
3 Mount Sinai Hospital, Samuel Lunenfeld Research Institute, Toronto,
Canada
4 Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of
Medicine, New York, USA
5 Departments of Pharmacology, Biochemistry and Molecular Biology, University of
Montreal, Montreal, Canada
Author for correspondence (e-mail:
trang.hoang{at}umontreal.ca)
Accepted 28 October 2003
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SUMMARY |
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Key words: VEGF, SCL, TAL1, Primitive erythropoiesis, Hematopoiesis, ES cell differentiation, Mouse
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Introduction |
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Gene-targeting experiments indicate a prerequisite role for vascular
endothelial growth factor A (VEGF)
(Ferrara and Henzel, 1989;
Gospodarowicz et al., 1989
)
signaling and for the basic helix-loop-helix (bHLH) transcription factor SCL
(also known as TAL1) (Begley et al.,
1989
; Finger et al.,
1989
; Chen et al.,
1990
) during the establishment of the hematopoietic system. VEGF
interacts with two tyrosine kinase receptors, Flt1
(Shibuya et al., 1990
) and
Flk1 (KDR Mouse Genome Informatics)
(Matthews et al., 1991
;
Millauer et al., 1993
;
Yamaguchi et al., 1993
). The
role of Flt1 during hematopoietic development is unclear because mice lacking
the tyrosine-kinase domain of Flt1 have no obvious hematopoietic defects
(Hiratsuka et al., 1998
). In
contrast, Flk1-deficient embryos die at midgestation (E8.5-9.5) because of the
absence of blood islands (Shalaby et al.,
1995
). When differentiated in vitro,
Flk1/ embryonic stem (ES) cells retain the
capacity to produce hematopoietic cells
(Hidaka et al., 1999
;
Schuh et al., 1999
),
suggesting that Flk1 is not involved in hematopoietic commitment per se. In
chimeras, Flk1/ cells fail to contribute to
primitive and definitive hematopoiesis
(Shalaby et al., 1997
).
Instead, they accumulate aberrantly on the surface of the amnion, which
indicates that VEGF might be involved in the migration of Flk1-positive
precursors from the mesoderm to sites of hematopoiesis, as reported for
Drosophia (Cho et al.,
2002
). As with Flk1/ embryos,
the loss of a single Vegf allele is lethal between E8.5 and E9.5
(Carmeliet et al., 1996
;
Ferrara et al., 1996
) because
of defects in blood island formation
(Damert et al., 2002
). This
reveals a unique, tight dose-dependent regulation of embryonic vessel and
hematopoietic development by VEGF.
SCL also plays a central role at the onset of hematopoiesis and
vasculogenesis. SCL is first co-expressed with Flk1 at E7.0 in cells of the
visceral mesoderm that are destined to generate blood islands. As blood
islands develop, SCL expression is maintained in primitive erythrocytes and at
low levels in endothelial cells, whereas Flk1 becomes restricted to vascular
cells (Shalaby et al., 1995;
Shalaby et al., 1997
;
Elefanty et al., 1999
;
Drake and Fleming, 2000
). Gene
targeting and chimera analyses reveal that SCL is required for the generation
of primitive and definitive hematopoietic lineages and for the remodeling of
yolk sac vasculature (Robb et al.,
1995
; Shivdasani et al.,
1995
; Robb et al.,
1996
; Porcher et al.,
1996
; Visvader et al.,
1998
).
Evidence is accumulating to indicate that SCL might function downstream of
VEGF/Flk1 signaling. First, SCL expression is subsequent to Flk1 in vivo
(Elefanty et al., 1999;
Drake and Fleming, 2000
) and
during the differentiation of ES cells in vitro
(Robertson et al., 2000
).
Importantly, SCL expression is not detected in
Flk1/ embryos
(Ema et al., 2003
). Secondly,
Flk1 and SCL are both required for blood island development
(Robb et al., 1995
;
Shalaby et al., 1995
;
Shivdasani et al., 1995
;
Visvader et al., 1998
).
Moreover, gain-of-function studies indicate that SCL might act at the level of
the putative, common hematopoietic and endothelial precursor, the
hemangioblast, to specify and promote hematopoietic and endothelial fates at
the expense of other mesoderm-derived tissues
(Gering et al., 1998
;
Mead et al., 1998
;
Mead et al., 2001
;
Ema et al., 2003
).
Interestingly, ES cell-derived hemangioblasts (also known as blast
colony-forming cells, or BL-CFCs) are found mostly in the Flk1/SCL
double-positive population (Chung et al.,
2002
) and require VEGF
(Kennedy et al., 1997
;
Choi et al., 1998
) and SCL
function (Faloon et al., 2000
;
Robertson et al., 2000
;
Ema et al., 2003
). Finally,
SCL can rescue the hematopoietic and vascular defect of the Zebrafish mutant
cloche (Liao et al.,
1998
), which acts upstream of Flk1
(Liao et al., 1997
), and allow
blast colony formation in the absence of Flk1 signaling in vitro
(Ema et al., 2003
). However,
it is not clear whether SCL rescues the multiple defects associated with Flk1
deficiency in vivo.
Hematopoietic cells have a finite life span in vitro and in vivo. When hematopoietic progenitors are plated in culture with the appropriate growth factors, they survive and first proliferate actively but eventually cease growth. It is not clear whether this growth arrest is determined intrinsically, or whether it can be influenced by environmental factors. Despite the importance of Flk1 signaling in hematopoiesis, it is not clear how VEGF/Flk1 regulates the development of hematopoietic cells. In the present study, we used cellular and genetic approaches to further define the role of VEGF and SCL at the onset of hematopoiesis.
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Materials and methods |
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Flow cytometry and antibodies
Yolk sacs were dissociated in 0.25% collagenase (Sigma-Aldrich) in PBS
supplemented with 20% FCS. Cells were first immunostained as described
(Herblot et al., 2000) using a
phycoerythrin-conjugated TER119 antibody (Pharmingen BD Biosciences). Cells
were then labeled with a fluorescein isothiocyanate-conjugated Annexin V
(Pharmingen) as described previously
(Krosl et al., 1998
). Included
was 7-amino actinomycin D (7-AAD, Calbiochem) to detect dead cells. Cells were
analyzed on a FACScalibur flow cytometer (Becton-Dickinson).
Histology and immunohistochemistry
Yolk sacs were fixed in 4% paraformaldehyde in PBS. Tissues were washed
with PBS and gelled in 2% agarose to facilitate transversal sectioning once
embedded in paraffin. Agarose embedding did not hinder the staining of
sections with dyes or antibodies. Samples were sectioned at 5 µm.
For immunohistochemistry, deparaffinized slides were placed in 1% SDS in PBS for 5 minutes then washed with water. Endogenous peroxydase activity was blocked with 1% H2O2, the fixative was quenched with 300 mM glycine and nonspecific binding was blocked with 10% horse serum (Sigma) in PBT (PBS supplemented with 0.2% tween-20). Sections were first incubated with a mouse antibody directed against KI67 (Pharmingen), overnight at 4°C. Slides were washed with PBT then incubated with a biotin-conjugated horse anti-mouse antibody (Vector Laboratories) followed by streptavidin-horseradish peroxydase (NEN), both incubated for 1 hour at room temperature. Positive cells were revealed with the peroxydase substrate 3,3'-diaminobenzidine (Sigma) and counterstained with methyl green.
Growth and differentiation of ES cells
Parental wild-type R1 (Nagy et al.,
1993), Vegf/ clones 36.7, 44.7
and 44.8 (Carmeliet et al.,
1996
) and the feeder-independent CCE
(Robertson et al., 1986
) ES
cell lines have been described previously. ES cells were maintained on
irradiated mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium
(Gibco) supplemented with 15% FCS (Gemini Bio-Products), 1000 U
ml1 leukemia inhibitory factor (LIF) and
1.5x104 M monothioglycerol (MTG, Sigma). Prior to
differentiation studies, feeder cells were diluted out following 3-4
sequential passages on gelatinized flasks.
Embryoid bodies (EBs) were generated as previously described
(Keller et al., 1993).
Briefly, dissociated ES cells were plated at a concentration of
0.3x104-1.0x104 ml1 into
Iscove's modified Dulbecco's medium (IMDM, Gibco) supplemented with 15% FCS
(Gibco), 2 mM glutamine (Gibco), 50 µg ml1 ascorbic acid
(Sigma), 5% protein-free hybridoma medium (PFHM II, Gibco) and
3x104 M MTG. When indicated, recombinant human
VEGF165 (Sigma) was added 3 days after the initiation of EB
differentiation (day 3). Day 7 EBs were dissociated into a single-cell
suspension using 0.25% trypsin, 1 mM EDTA (Gibco). The size of EBs and
primitive erythroid colonies (EryP) was assessed using imaging
software (Northern Eclipse, Empix Imaging). Blast colonies were generated in
methylcellulose cultures containing 10% FCS, 5 ng ml1
interleukin 6 (IL-6) and 20% D4T endothelial conditioned medium as previously
described (Kennedy et al.,
1997
). When indicated, 5 ng ml-1 VEGF was added to
cultures.
Hematopoietic colony assays and growth factors
Hematopoietic colonies were generated by plating either dissociated day 7
EB cells at 4x105 ml1 or half of
dissociated E8.5 yolk sac in IMDM containing 1% methylcellulose, 10% FCS
(Gibco), 5% PFHM II, 200 µgml1 transferrin, 100 ng
ml1 KL, 2 U ml1 EPO,
5ngml1 IL-6, 5 ng ml1 IL-3, 5 ng
ml1 M-CSF, 30 ng ml1 G-CSF (Amgen),
1ngml1 LIF, 5ngml1 VEGF and
1.5x104 M MTG. KL, EPO, IL-6, IL-3, M-CSF, and LIF
were derived from media conditioned by COS cells transfected with
corresponding expression vectors.
Gene expression
Representative amplification of total cDNA was carried out as described
previously (Sauvageau et al.,
1994). Amplified cDNA was resolved on 1.2% agarose gels and
transferred to nylon membranes (Pall Corporation) for hybridization. ßH1:
269-bp fragment amplified by PCR using forward primer
5'-TTGTTACAGCTCCTGGGCA-3' and reverse primer
5'-CCCAAAAAGTCAATGTTATT-3'. ßmajor: 135-bp fragment
immediately upstream of the polyA tail. Gata1, 443-bp fragment was
amplified by PCR using forward primer 5'-GGAGACAGGATCTTCTGTAG-3'
and reverse primer 5'-CATGCTCCACTTGACACTGA-3'. Scl:
438-bp fragment was prepared by PCR using forward primer
5'-CATAACCACAGAGAGAATCCC-3' and reverse primer
5'-ACACTATCATCACCACACTGG-3'
(Hoang et al., 1996
).
Flk1: 944 bp 3' HincII fragment. Ribosomal
L32: genomic 1.6 kb SacI fragment encompassing the final
exon. The L32 probe was kindly provided by Dr N. Iscove and the
ßH1 probe by Dr G. Sauvageau. The hybridation signals were analyzed on a
PhosphorImager apparatus (Molecular Dynamics).
Quantification of Scl mRNA from Scltg yolk
sacs was accomplished using the Quantitect SYBR Green PCR kit (Quiagen),
performed on a MX4000 apparatus (Stratagene) following the manufacturer's
instructions. Briefly, cDNA was generated as described previously
(Herblot et al., 2000),
normalized for equal S16 levels and either endogenous mouse or
transgenic human Scl levels quantified using standard curves
determined with known molar amounts of either mouse or human Scl
templates, respectively. S16: forward primer,
5'-AGGAGCGATTTGCTGGTGTG-3'; reverse primer,
5'-GCTACCAGGGCCTTTGAGATG-3'. Mouse Scl: forward primer,
5'-GGGCAGTTGATGTGTTTGTGTCA-3'; reverse primer,
5'-GCCCAGCCCCTTTAGAAACTTTC-3'. Human Scl: forward primer,
5'-TCCCTATGTTCACCACCAAC-3'; reverse primer,
5'-GATGTGTGGGGATCAGCTT-3'.
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Results |
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To better characterize the effect of VEGF, EBs were analyzed individually. Although hemoglobinization occured in most EBs in the absence of VEGF, few if any of these EBs gave rise to hematopoietic colonies upon replating (Fig. 2A). In contrast, in the presence of VEGF, 15 out of 25 EBs gave rise to primitive erythroid colonies, with a mean of 10 EryP per EB. The increase in EryP cannot be accounted for on the basis of size expansion, because the average number of cells per EB was increased only by 1.6 fold (data not shown). Thus, our observations indicate that VEGF significantly enhances the erythroid potential of ES cells.
|
VEGF influences the developmental potential of primitive erythroid colonies
Results shown in Fig. 2
indicate that the expansion of the primitive erythroid compartment in response
to VEGF might be attributable to an increase in the number of erythroid
progenitors within each EB. In addition, it is possible that each erythroid
progenitor exhibits an increased proliferation potential and extended life
span. Therefore, we assessed the cellularity of primitive erythroid colonies
derived from Vegf/ EBs that developed in
either the presence or absence of VEGF by integrating the area of individual
colonies using imaging software. The distribution of colony size revealed that
primitive erythroid precursors that developed in the presence of VEGF gave
rise to larger EryP. Indeed, data shown in
Fig. 3A indicate that
EryP isolated from VEGF treated EBs are larger than colonies
derived from untreated EBs, as seen by a shift to the right of their size
distribution (P<0.003).
|
Because primitive erythroid precursors do not express Flk1
(Drake and Fleming, 2000),
their expansion on addition of VEGF could be caused by an effect on earlier
developmental stages. We therefore determined the effect of VEGF on BL-CFCs,
which represent the earliest committed hematopoietic precursors and express
Flk1 (Kennedy et al.,
1997
; Chung et al.,
2002
). ES cells were allowed to differentiate into either day 3 or
day 3.5 EBs and replated into hematopoiesis either with or without VEGF. As
shown in Fig. 3C, VEGF strongly
increased the number of blast colonies at both time points. These results
imply that the expansion of the primitive erythroid compartment by VEGF might
be attributable to an earlier effect on hemangioblast-like cells.
Vegflo homozygous mice die by E9.5
The role of VEGF in vivo was defined further through reducing the VEGF dose
in Vegf low (Vegflo) hypomorph mice. These mice carry an
internal ribosomal entry site (IRES)-lacZ insertion immediately downstream of
the Vegf gene STOP codon, which disrupts the post-transcriptional
processing of Vegf mRNA and renders a functionally hypomorph allele
(Damert et al., 2002). Embryos
dissected at E8.0 were viable and occurred at the frequency expected for
Vegf+/+, Vegflo/+ and
Vegflo/lo, indicating that lethality occurs later
(Table 1). When embryos
carrying the Vegflo allele were analyzed between E8.5-E9.5
(8-26 somite pairs), all Vegflo/lo embryos had <20
somite pairs, whereas a significant proportion of wild-type and heterozygous
embryos had >20 somite pairs. This analysis revealed that the mutation is
homozygous lethal by E9.5 (n=220). Although heterozygous
Vegflo/+ embryos had no obvious abnormalities,
morphological and histological analysis of Vegflo/lo
littermates showed similar defects to those seen in
Vegf+/ embryos
(Carmeliet et al., 1996
;
Ferrara et al., 1996
), that is
a reduced dorsal aorta lumen, disorganized yolk sac vasculature and reduced
numbers of blood cells in both the embryo proper and blood islands
(Damert et al., 2002
) (data not
shown).
|
|
VEGF is essential for the survival of primitive erythrocytes
The reduced number of primitive erythrocytes in
Vegflo/lo hypomorph embryos could be caused by either
decreased proliferation or increased apoptotic death as hematopoietic cells
undergo apoptosis in the absence of appropriate growth factors. Therefore, we
assessed the effect of reduced VEGF activity on the survival and proliferation
of differentiating primitive erythrocytes. Yolk sac erythroid cells were
isolated between E9.0-E9.5 (12-26 somite pairs) and stained with TER119 and
Annexin V, which recognize membrane phosphatidylserine residues that are
exposed during the initial stages of apoptotic cell death. Consistent with
colony assays and gene expression analysis, the frequency of TER119-positive
cells correlated with the presence of either one or two hypomorph
Vegflo alleles and was lowest in
Vegflo/lo embryos (Fig.
5A, Table 2).
Furthermore, there was a direct correlation between VEGF activity and the
survival of primitive erythrocytes. The proportion of TER119-positive cells
that also stained for Annexin V was 2.3-fold and 4.8-fold higher in
heterozygous Vegflo/+ and homozygous
Vegflo/lo embryos, respectively, compared to wild-type
littermates. Interestingly, in heterozygous embryos, the level of apoptosis
within the TER119-positive population increased as the embryos matured. The
frequency of TER119-positive cells undergoing apoptosis was 19.7±7.7%
for embryos between 18-21 somite pairs (n=3) and 48.6±7.3%
between 22-25 somite pairs (n=3). These observations concur with the
analysis of viability shown in Table
1, and indicate a requirement for high VEGF activity after 20
somite pairs. Finally, there was a significant increase in the level of
apoptosis in TER119-negative cells in Vegflo/lo embryos
that was not observed in heterozygous and wild-type embryos, which was
possibly caused by an increase in apoptosis in non-erythroid cells or by the
loss of TER119 surface marker as erythroid cells die. Taken together, these
results indicate that high VEGF activity is required for the survival of
primitive erythrocytes.
|
|
SCL interacts with VEGF to suppress apoptosis in primitive erythroid cells
The induction of Scl by VEGF in vitro and in vivo could either be
a cause or a consequence of increased hematopoiesis. To distinguish between
these two possibilities, we asked whether elevation of SCL could substitute
for defective VEGF activity in Vegflo/lo embryos. To this
end, heterozygous Vegflo/+ mice were bred with
Scl transgenic mice that consitutively express Scl under the
control of the Sil (Scl interrupting locus) promoter
(Aplan et al., 1997) to
generate compound-heterozygote Vegflo/+Scltg
mice, that were then crossed to produce
Vegflo/loScltg embryos. In wild-type embryos,
the Sil-Scl transgenic cassette allows 14.5-fold higher level of
Scl in the yolk sac compared to non-transgenic littermates
(Fig. 6A). Analysis of TER119
labeling indicated a modest increase in TER119-positive cells when the
Scl transgene was introduced in a Vegflo/+ and
Vegflo/lo background, but not in wild-type embryos
(Table 2). Annexin V labeling
revealed that the survival of TER119-positive cells was dependent on the
number of functional Vegf alleles. Thus, apoptotic death was 70%, 35%
and 15% of TER119-positive cells, in Vegflo/lo,
Vegflo/+ and Vegf+/+ embryos, respectively
(Fig. 6B). Strikingly, the
Scl transgene reduced cell death by half in
Vegflo/lo and Vegflo/+ embryos,
indicating an important anti-apoptotic function for the SCL transcription
factor. To assess whether the suppression of cell death by SCL and VEGF
occurred through parallel pathways or the same pathway, we examined apoptosis
in Vegf+/+ embryos. As shown in
Fig. 6B, the anti-apoptotic
effect of SCL was not additive to that of VEGF. We therefore surmise that SCL
and VEGF operate within the same genetic pathway to determine the output in
primitive erythroid cells. Consistent with a partial restoration of primitive
erythropoiesis (Table 2),
Gata1 and ßH1 transcripts
(Fig. 6C,D) were present at low
levels in the yolk sac of Vegflo/loScltg
embryos, whereas they were below the limit of detection in
Vegflo/lo embryos. Together, our observations indicate
that elevating Scl levels suppresses apoptosis and allows an
expansion of Flk1-positive cells.
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Discussion |
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Different VEGF thresholds are required during hematopoietic development
Knockout and chimera studies have linked VEGF function to the migration of
hematopoietic precursors from the mesoderm to the yolk sac and for the
generation of blood islands (Shalaby et
al., 1995; Shalaby et al.,
1997
; Carmeliet et al.,
1996
; Ferrara et al.,
1996
, Damert et al.,
2002
). We used Vegflo hypomorph mice,
developed by Damert et al. (Damert et al.,
2002
), to further define the effect of VEGF dose on cell-fate
decisions involved with the development of the hematopoietic system. Although
heterozygous Vegflo/+ embryos are viable, homozygous
Vegflo/lo littermates die by E9.5 because of hematopoietic
and vascular defects that are similar to those of
Vegf+/ embryos. From this, we infer that each
Vegflo allele provides <50% activity of a wild-type
allele. Thus, by varying the number of Vegflo alleles, we
compared hematopoietic development in embryos exposed to a range of VEGF
activity; homozygous Vegflo/lo embryos provided
50%
and heterozygous Vegflo/+ littermates, 75% of wild-type
VEGF activity.
In Vegflo/lo embryos, in which VEGF activity is
presumed to be 50% of wild type, Flk1-positive mesodermal precursors reach the
yolk sac but are severely compromised in their capacity to expand and
differentiate into primitive erythroid cells. Indeed, Flk1 expression is
detected clearly in Vegflo/lo yolk sacs, but it is
diminished in comparison to heterozygous and wild-type littermates because of
the substantial loss of Flt1/Flk1-positive mature endothelial cells
(Damert et al., 2002). However,
in Vegflo/+ littermates, when VEGF activity is raised to
higher levels, the embryos survive, thus setting a threshold for the
development of blood islands and for the expansion of the primitive erythroid
compartment. Moreover, we observed a direct relationship between the level of
VEGF activity and the number of primitive erythroid precursors per yolk sac,
indicating a tight dependence of the primitive erythroid lineage on the number
of functional VEGF alleles. In contrast to the dose-dependent requirement for
sustained VEGF activity during primitive erythropoiesis, inactivation of both
Vegf alleles was needed to abrogate the survival of adult
hematopoietic stem cells (Gerber et al.,
2002
). Consistent with studies in vivo, addition of VEGF to
differentiating Vegf/ ES cells in vitro
increased the frequency of primitive erythroid precursors in a dose-dependent
manner. This effect of VEGF on primitive erythropoiesis was observed using
three independent Vegf/ clones (36.7, 44.7
and 44.8) and the parental R1 ES cells. However, we did not observe an
increase in EryP by VEGF using the feeder-independent CCE line, as
previously described (Kabrun et al.,
1997
). CCE cells are efficient for hematopoietic differentiation,
possibly because of a higher level of endogenous VEGF secretion. Furthermore,
addition of VEGF to differentiating ES cells stimulated the clonal expansion
of each precursor, giving rise to more primitive erythrocytes per colony and
extended their life span in culture. Although not proven directly, extension
of the life span of primitive erythrocytes by VEGF may be interpreted as a
delay in their senescence. Mechanisms that underlie senescence are only
beginning to emerge, and point to the importance of telomere erosion,
cell-cycle control and growth conditions (reviewed by
Rubin, 1998
;
Sherr and DePinho, 2000
). Our
results raise the question whether growth factors may also be involved in the
senescence process, possibly by shaping the developmental potential of early
progenitors long before the growth arrest of their progeny is observed in
culture.
Our results indicate that VEGF enhances primitive erythropoiesis, but
primitive erythrocytes do not express Flk1 and Flt1
(Shalaby et al., 1995;
Shalaby et al., 1997
;
Fong et al., 1996
;
Drake and Fleming, 2000
).
There may be several possibilities. First, it is possible that a third, as yet
unidentified, VEGF receptor is expressed on primitive erythrocytes. Second,
VEGF might stimulate primitive erythropoiesis indirectly, through the
secretion of a secondary hematopoietic growth factor from Flk1-positive
vascular cells. Third, it is conceivable that VEGF affects the developmental
potential of an earlier Flk1-positive precursor by promoting a hematopoietic
fate. Although we cannot exclude the first and second possibilities, we favor
the third. Given that reduced VEGF activity also affects endothelial
development (Damert et al.,
2002
), we speculate that the reduction in the number of primitive
erythroid precursors in Vegflo/lo embryos is caused, for
the most part, by the inability of Flk1-positive putative hemangioblasts to
expand and differentiate into blood islands. Similarly, when VEGF is added to
differentiating EBs, enhancement of primitive erythropoiesis might also occur
at the hemangioblast stage. Indeed, ES cell-derived hemangioblasts, BL-CFCs,
appear transiently after 3-4 days of differentiation
(Kennedy et al., 1997
;
Choi et al., 1998
) at the time
when Flk1 is first detected (Kabrun et
al., 1997
) and at the time when we add VEGF to our cultures. In
agreement with this interpretation, VEGF strongly enhanced the number of
BL-CFCs isolated from day 3 and day 3.5 EBs. Thus, the increase in the number
of EryP in day 7 EBs might, therefore, result from an in situ
expansion of BL-CFCs in day 3-4 EBs. It is noteworthy that the only difference
between VEGF-treated and control cultures is the presence of VEGF during days
3-7 of EB differentiation. The growth factor cocktail for the hematopoietic
colony assay is identical and contains VEGF. Thus, the effect of VEGF must
occur at the earliest stages of hematopoietic commitment.
VEGF is essential for the survival of primitive erythrocytes: partial rescue by SCL
VEGF has an established role in endothelial cell function, favoring the
proliferation and survival of endothelial cells during development and in
adults (Ferrara et al., 2003).
VEGF is essential for the survival of hematopoietic stem cells, through Flk1
and possibly Flt1 signaling (Gerber et
al., 2002
), although the effect of VEGF during primitive
erythropoiesis has not yet been defined. Analysis of
Vegflo hypomorph embryos revealed a direct relationship
between the number of Vegflo alleles and the frequency of
apoptotic, TER119-positive, primitive erythroid cells, while reduced VEGF
activity had little effect on the proliferation of the same cells. Strikingly,
overexpression of SCL, using a Scl transgene under the control of the
ubiquitous Sil promoter, partially alleviated the apoptosis of
primitive erythrocytes associated with the Vegflo allele,
which correlated with an increase in TER119 staining and ßH1 expression
in individual yolk sacs. We have shown previously that SCL functions
downstream of the Flk1-related tyrosine kinase c-kit to promote the survival
of definitive hematopoietic precursors
(Krosl et al., 1998
)
(unpublished data). Simlarly, in this study, we provide evidence that
VEGF/Flk1 signaling enhances primitive erythropoiesis by promoting the
survival of primitive erythrocytes through the anti-apoptotic function of
SCL.
To date, few transcription factors have been identified that determine cell
fate. For example, the expression of MyoD, a member of the bHLH
family, is sufficient to induce muscle formation
(Davis et al., 1987). Several
groups have shown that SCL can specify and promote hematopoietic and vascular
fates at the expense of other mesodermal tissues
(Gering et al., 1998
;
Mead et al., 1998
;
Mead et al., 2001
). However,
it remains unclear how SCL potentiates hematopoietic and vascular fates. Ema
et al. (Ema et al., 2003
) have
shown recently that SCL favors the endothelial lineage at the expense of
smooth muscle in a VEGF dependent process. They have also shown that SCL acts
downstream of Flk1, at the hemangioblast level, to rescue hematopoietic and
vascular defects in vitro. Our work indicates that enhancement of the
hematopoietic fate might result, at least in part, from the increased survival
of primitive erythrocytes as a result of VEGF/Flk1-induced Scl
expression.
Taken together, our data indicate that during the establishment of the hematopoietic system, in addition to guiding the migration of hematopoietic and endothelial precursors, VEGF enhances the hematopoietic fate by expanding the primitive erythroid compartment and potentiating the survival of primitive erythroid cells through SCL function in hemangioblasts.
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ACKNOWLEDGMENTS |
---|
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Footnotes |
---|
Present address: Laboratoire de génétique et physiologie du
développement, IBDM-campus de Luminy, Marseille, France
Present address: Institute of Research in Immunovirology and Cancer (IRIC),
University of Montreal, PO Box 6128, Downtown Station, Montreal, Quebec H3C
3J7, Canada
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