1 Howard Hughes Medical Institute and Abramson Family Cancer Research Institute,
University of Pennsylvania School of Medicine, Philadelphia, PA 19104,
USA
2 Abramson Family Cancer Research Institute, University of Pennsylvania School
of Medicine, Philadelphia, PA 19104, USA
3 Department of Immunology, Medical Faculty/University Clinics, 89070 Ulm,
Germany
4 Carl C, Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai
School of Medicine, New York, NY 10029, USA
* Author for correspondence (e-mail: celeste2{at}mail.med.upenn.edu)
Accepted 15 June 2004
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SUMMARY |
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Key words: HIF, ARNT, Hypoxia, Hemangioblast, Mesoderm
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Introduction |
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HIF modulates the transcription of these genes and is globally activated
during embryonic development in organs that naturally experience an
O2 gradient (Iyer et al.,
1998; Mitchell and Yochim,
1968
; Rodesch et al.,
1992
). HIF is a member of the basic helix-loop-helix (bHLH)-PAS
family of proteins that regulate many essential processes, including
O2 homeostasis, circadian rhythms, neurogenesis and toxin
metabolism (Gu et al., 2000
;
Wang et al., 1995
). Members of
the HIF subfamily of bHLH-PAS proteins heterodimerize to form transcriptional
complexes that induce gene expression by binding an
50-bp Hypoxia
Response Element (HRE) (Semenza,
1998
; Semenza,
1999
; Semenza et al.,
1991
). Under normoxic conditions, the HIF
subunits
HIF1
, HIF2
(EPAS) and HIF3
are targeted for proteasome
degradation by the von Hippel-Lindau (VHL) protein
(Cockman et al., 2000
;
Maxwell et al., 1999
;
Ohh et al., 2000
). However,
under hypoxic conditions, HIF
subunits are stabilized, translocate to
the nucleus, and dimerize with the ß-subunits ARNT (aryl hydrocarbon
receptor nuclear translocator) or ARNT2
(Semenza, 1999
). Null
mutations in either HIF1
or ARNT lead to midgestational lethality of
embryos, with phenotypes that include defects in the vasculature, blood,
placenta and heart (Adelman et al.,
2000
; Adelman et al.,
1999
; Iyer et al.,
1998
; Kotch et al.,
1999
; Maltepe et al.,
1997
; Ryan et al.,
1998
). Thus, improper responses to low O2 in the embryo
can lead to lesions in multiple aspects of cardiovascular development.
Our original characterization of Arnt/
embryos demonstrated a vascular remodeling defect in the extraembryonic yolk
sac (Maltepe et al., 1997).
Further clonogenic analysis of Arnt/ yolk
sacs revealed a defect in the generation of hematopoietic progenitors
(Adelman et al., 1999
). As the
yolk sac endothelial cell defect also extends to embryonic tissues
(Keith et al., 2001
;
Maltepe et al., 1997
), we
postulated that embryonic lethality could be the result of a defect in an
early progenitor of the cardiovascular system. Because embryonic hematopoiesis
is spatially and temporally linked to endothelial cell development,
Arnt may independently be required for the differentiation of both
lineages, or alternatively be essential for the production of `hemangioblast'
progenitors.
The notion that endothelial and hematopoietic cells are derived from a
common hemangioblastic precursor is based on observations that these lineages
emerge simultaneously and in proximity to each other during organogenesis
(Sabin, 1920):
extraembryonically in the yolk sac blood islands, and intraembryonically in
the aorta-gonad-mesonephros (AGM) (Haar
and Ackerman, 1971
; Medvinsky
and Dzierzak, 1996
; Muller et
al., 1994
). Endothelial and hematopoietic cells also share
expression of multiple genes (reviewed by
Choi, 1998
). FLK1+
cells are first detected in blood island mesodermal aggregates that contribute
to the extraembryonic vasculature of the yolk sacs in 7.0 dpc mouse embryos
(Choi, 1998
;
Choi et al., 1998
;
Dumont et al., 1995
). More
recently, an intraembryonic source of potential hemangioblast cells has been
identified in the endothelium of the dorsal aorta
(de Bruijn et al., 2002
;
North et al., 2002
).
Although attempts to isolate hemangioblasts from embryos have not been
successful, a morphologically distinct cell or `blast colony-forming cell'
(BL-CFC) has been described in embryonic stem (ES) cell-derived `embryoid
bodies' (EBs) (Choi, 1998;
Choi et al., 1998
;
Kennedy et al., 1997
). BL-CFCs
are likely to represent hemangioblasts, as they exclusively produce both
endothelial and blood cells in vitro. Significantly, early embryonic
development can be mimicked by in vitro differentiation of ES cells into EBs
composed of all three germ layers (Keller
et al., 1993
; Keller,
1995
). The differentiating ES cell masses are an advantageous
model system in which mutant ES cells can be synchronized, manipulated and
analyzed for their production of various cell lineages. Using this assay
system, we have previously demonstrated that hypoxia increases hematopoietic
precursor numbers (Adelman et al.,
1999
). These in vitro assays confirmed that
Arnt/ ES cells generate significantly fewer
numbers of hematopoietic progenitors, consistent with results obtained from
Arnt/ yolk sacs
(Adelman et al., 1999
). The
putative hemangioblast, or BL-CFC, appears transiently within the mesoderm of
differentiating EBs. Indeed, mesodermal progenitors that express Brachyury, a
T-box transcription factor, differentiate into hemangioblasts, and subsequent
hematopoietic and endothelial lineages
(Fehling et al., 2003
). The EB
differentiation system has facilitated the elucidation of cell-intrinsic
factors required for the generation of hemangioblasts, including the VEGF
receptor FLK1, the bFGF receptor FGFR1, the Ephrin receptor EPHB4, and the
transcription factors SCL (TAL1) and RUNX1 (AML1)
(Ema et al., 2003
;
Faloon et al., 2000
;
Fehling et al., 2003
;
Lacaud et al., 2002
;
Robertson et al., 2000
;
Wang et al., 2004
).
Given the observed hematopoietic and endothelial defects in Arnt/ embryos, we investigated the role of hypoxia in the commitment of mesoderm to the hemangioblast lineage. We report here that hypoxia indeed promotes the generation of hemangioblasts from ES cells. In fact, hypoxia induces earlier expression of Brachyury, Flk1 and Bmp4 in EBs. Interestingly, both Arnt and Vegf mutant ES cells are deficient in the generation of BL-CFCs. Moreover, we establish that the hemangioblast defect in Arnt/ cells is not cell-intrinsic, although multiple growth factors (including VEGF and bFGF) are not sufficient to rescue the phenotype. These findings suggest that hypoxia-mediated generation of mesoderm, and of blood and vascular progenitor cells, is crucial for early embryonic development.
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Materials and methods |
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Methylcellulose colony assays
EBs were disaggregated in 0.25% Trypsin-EDTA (Invitrogen), and further
dissociated with a 21-gauge needle. 5x104 cells were replated
in triplicate in 1% methylcellulose medium (H4100, SCT), supplemented as
described (Choi et al., 1998;
Kennedy et al., 1997
). For
mixing conditions, equal numbers of Arnt+/+ and
Arnt/ ES cells were co-cultured during EB
differentiation. Disaggregated EBs were replated in methylcellulose as above.
One set of triplicate cultures was treated with 0.4 mg/ml of G418. X-gal
staining for the expression of ß-galactosidase was performed on
individually picked colonies that were fixed in 5% paraformaldehyde for 10
minutes, washed in PBS, and stained as previously described
(Schuh et al., 1999
).
Matrigel cultures
Individual colonies were transferred to MatrigelTM (Collaborative
Research)-coated 96-well plates and cultured for 10 days, as described
(Choi et al., 1998). All
cytokines were purchased from R&D Systems, except rhEpo (Amgen) and ECGS
(Collaborative Bioresearch). DiI-acetylated low density lipoprotein
(DiI-Ac-LDL; Biomedical Technologies) endothelial uptake was performed by
adding 10 µg/ml to Matrigel cultures for 4 hours at 37°C. Cultures were
fixed with 4% paraformaldehyde, washed, and observed by fluorescence
microscopy using a rhodamine filter.
Gene expression analysis
RNA was isolated by the TRIzol method (Invitrogen). After treatment with
DNaseI (Invitrogen), reverse transcription was performed with Superscript-II
reverse transcriptase (Invitrogen), using oligo dT primers (Promega). PCR
reactions were performed as previously described
(Schuh et al., 1999), with
sequence-specific primers (10 pmol per reaction) published by Roberston or
Schuh et al. (Roberston, 2000; Schuh et
al., 1999
) for ß-actin. Radioactive PCR was performed by
adding 0.01 µl of
-dCTP to PCR reactions, which were separated on
acrylamide gels, dried, and visualized by PhosphoImager analysis.
Real-time detection PCR (RTD-PCR) was performed as previously described
(Seagroves et al., 2003). PCR
reactions were performed using default cycling parameters of the ABI Prism
7900HT Sequence Detector. Reactions were carried out in a 20 µl reaction,
with 2xTaqman Master Mix (ABI) and the following primers:
Each target gene was normalized to 18S RNA (ABI, catalog number 4308329)
for each sample using the Ct method (threshhold values)
(Muller et al., 2002
).
Relative mRNA levels were then compared, at each time point, to wild-type
normoxic samples, which were normalized to a value of one, and data was
expressed as fold induction of mRNA.
Flow cytometry
After 1.5 to 4 days of differentiation, EBs were disaggregated as
previously reported (Faloon et al.,
2000). Cells were incubated in 1:100 blocking buffer
Fc
III/II receptor (Pharmingen) for 20 minutes at 4°C. After washing
in 1% BSA/PBS, cells were incubated with PE-conjugated FLK1 [Avas12
1
(Pharmingen)] at 1:100 in wash buffer for 20 minutes at 4°C, washed and
visualized by FACS Vantage (Becton Dickinson). Results were analyzed by Flo-Jo
(Tree-Star).
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Results |
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BL-CFCs appeared as loosely adherent cell clusters in methylcellulose (Fig. 1A,G'), and when replated on matrigel yielded both adherent endothelial cells that formed characteristic tube-like structures and non-adherent hematopoietic cells (Fig. 1A,H'-I'). Subsequent differentiation of BL-CFCs confirmed that the adherent cells were endothelial cells, based on their uptake of fluorescent DiI-Ac-LDL (Fig. 1B). We further confirmed the BL-CFC phenotype by demonstrating the absence of early markers (Bry, Rex1) and the presence of endothelial/hematopoietic markers (Gata1, Scl) in both Arnt+/+ and Arnt/ cultures (Fig. 1C). These replating studies reveal that BL-CFC colonies obtained from Arnt+/+ and Arnt/ EBs have the potential to generate both hematopoietic and endothelial cells.
ARNT is required for appropriate generation of hemangioblast colonies
Although both Arnt+/+ and
Arnt/ EB cultures are capable of generating
functional BL-CFCs in vitro, hematopoietic and endothelial defects observed in
Arnt/ embryos may arise from reduced numbers
of functional hemangioblasts. To assess the quantitative effect of the
Arnt mutation on hemangioblast formation,
Arnt/ ES cells were analyzed for their
capacity to generate appropriate numbers of BL-CFCs.
Arnt+/+ and Arnt/ cells
were differentiated into EBs for 3 days and assayed for the development of
BL-CFCs. Of note, five independent Arnt/
clones from two independent Arnt+/ ES cell lines
generated significantly fewer BL-CFCs compared with
Arnt+/+ controls (P values ranging from 0.0117 to
0.03; Fig. 2A). By contrast,
Arnt/ cultures generated a higher proportion
of 2° EBs and Trans-CFCs in methylcellulose when compared with wild-type
cultures (Fig. 2B and data not
shown), suggesting proper mesoderm formation but an arrest in subsequent
differentiation into the hemangioblasts.
|
One explanation for the unexpected lack of a hypoxic stimulation of BL-CFCs
may be that mesoderm differentiation is influenced by low O2
conditions, hastening the kinetics of hemangioblast development. Although the
peak for BL-CFC generation is at 3.5 days of differentiation, the presence of
BL-CFCs is brief during EB differentiation
(Kennedy et al., 1997;
Robertson et al., 2000
).
Kinetic analysis of Trans-CFCs and BL-CFCs previously revealed that the
highest numbers of transitional colonies appear one day earlier than blast
colonies (Robertson et al.,
2000
). To delineate the kinetics of early progenitor colony
formation during EB differentiation, we performed differentiation time courses
of 1.5-4 days under normoxic and hypoxic conditions. Sample data from
experiments performed for 2 to 3.5 days are represented in
Fig. 2B,C. First, the number of
Trans-CFCs in Arnt+/+ cultures was higher at 2 and 2.5
days of differentiation for normoxic and hypoxic conditions than at day 3.
Interestingly, hypoxia increased Trans-CFC numbers on day 2 of differentiation
in Arnt+/+ cultures (P=0.008,
Fig. 2B). Second, under
normoxic conditions, wild-type ES cells exhibited an increase in BL-CFC
numbers between days 2 and 3.5. Of note, 2-2.5 days of hypoxic differentiation
further increased the generation of BL-CFCs compared with normoxia (day 2,
P=0.001; day 2.5, P=0.0013;
Fig. 2B,C). However, hypoxia
decreased BL-CFC numbers by day 3 of EB differentiation
(Fig. 2B,C). These results
suggest that low O2 not only influences the number of BL-CFCs
formed, but also alters the kinetics of EB differentiation into transitory
BL-CFCs (Fig. 2C). Conversely,
hypoxia did not greatly influence Arnt/
Trans- or BL-CFC colony numbers (Fig.
2B). When compared with Arnt+/+ cells, the
total numbers of Trans-CFCs were higher for
Arnt/ cells under normoxic conditions, and
they were unaffected by hypoxia (Fig.
2B, see also Fig.
5A). Therefore, it appears that
Arnt/ cells undergo a developmental arrest
and are blocked at the transitional stage. In all assays performed, an
increase in BL-CFCs was consistently observed in hypoxic
Arnt+/+ cultures, although the peak time of induction
varied by 12 hours between experiments. The kinetic experiments suggest that
`physiological' hypoxia encountered during embryogenesis contributes to the
proper and timely development of hematopoietic/endothelial progenitors, and is
dependent upon ARNT.
|
|
To complement the kinetic analysis of FLK1+ cell and
hemangioblast development, semi-quantitative RT-PCR was performed on EB
cultures at day 0 to 3.5 of differentiation
(Fig. 4A). Flk1
transcripts were amplified in hypoxic Arnt+/+ cultures one
day earlier than normoxic cultures, based on RT-PCR analysis
(Fig. 4A). Similar results were
obtained for Bry and Bmp4, a growth factor required for
extraembryonic mesoderm formation (Winnier
et al., 1995). Interestingly,
Arnt/ EB cultures expressed low levels of
Flk1, Bry and Bmp4 under normoxic and hypoxic conditions. By
contrast, Rex1 and Bmp2 expression was not affected by
O2 or by the presence of ARNT
(Fig. 4A).
|
Exogenous growth factors fail to rescue the Arnt/ FLK1 and hemangioblast defects
VEGF addition to methylcellulose cultures promotes the growth of BL-CFCs
(Kennedy et al., 1997;
Robertson et al., 2000
). If
omitted, the resulting colonies retain a more transitional phenotype.
Presently, the data demonstrate hypoxic upregulation of Vegf mRNA in
day 2 and 3 EBs (Fig. 4B).
Because HIF is an important transcriptional regulator of Vegf,
hemangioblast production by Vegf mutant ES cells was examined.
Interestingly, Vegf/ EBs were deficient in
generating Trans-CFCs and BL-CFCs when compared with wild-type cells
(Fig. 5A, normoxic conditions),
suggesting that VEGF is crucial for their production. Moreover, VEGF levels
are important: Vegf+/ EBs were also deficient in
generating BL-CFCs, although they produced increased numbers of Trans-CFCs, as
noted for Arnt/ EBs. In contrast to
Arnt/ EBs, hypoxia increased BL-CFC numbers
in Vegf+/ and
Vegf/ EB cultures
(Fig. 5A). Owing to the
transient nature of BL-CFCs, fewer BL-CFCs were generated by hypoxic
Arnt+/+ EBs on 3 day (see
Fig. 2B and
Fig. 5A).
As Vegf mutant cells were deficient in proper hemangioblast
production, and exogenous VEGF rescued the hematopoietic progenitor defect in
day 9 Arnt/ EB cultures
(Adelman et al., 1999), VEGF
was added to differentiating EBs in an attempt to `rescue' the hemangioblast
defect. In Arnt+/+ cultures, 5-10 ng/ml of VEGF
significantly increased the number of BL-CFCs as early as 1.5 days of
differentiation (Fig. 5B). Not
surprisingly, Vegf mutant cultures generated equivalent numbers of
BL-CFCs to wild-type cultures upon the addition of exogenous VEGF (data not
shown). However, VEGF did not significantly increase BL-CFCs in
Arnt/ cultures
(Fig. 5B). Interestingly, a new
colony type was generated in VEGF-treated
Arnt/ cultures that may represent a
degenerate progenitor colony with endothelial characteristics, as this colony
retains early markers (Bry, Rex1) based on RT-PCR analysis (see Fig.
S1 at
http://dev.biologists.org/cgi/content/full/131/18/4623/DC1).
Although FLK1 is necessary for proper BL-CFC generation
(Schuh et al., 1999), its
expression may require independent conditions from those required for BL-CFC
production. In contrast to Arnt/ EBs,
Vegf+/ and Vegf/
cultures generated significant numbers of FLK1+ cells
(Fig. 5C). These results
suggest an independent requirement for VEGF in the production of BL-CFCs,
distinct from the production of FLK1+ progenitors. VEGF was not
sufficient to fully rescue the Arnt/
FLK1+ cell or the BL-CFC defects (data not shown,
Fig. 5B). Therefore, bFGF was
also added, based on its ability to induce FLK1 surface expression in
differentiating EBs (Faloon et al.,
2000
). The addition of these growth factors did not significantly
increase FLK1+ cell numbers in either Arnt+/+
or Arnt/ EB cultures, but did enhance FLK1
expression in Vegf+/ and
Vegf/ cultures
(Fig. 5C). Although 2.75 days
of hypoxia effectively stimulated the production of FLK1+ cells in
Arnt+/+ cultures (Fig.
5C), FLK1 expression at 2.75 days in both normoxic and hypoxic
conditions was reduced when compared with levels detected at later times
(Fig. 3A).
FLK1 expression has been used as a hemangioblast marker as the presence of
FLK1 is required for proper BL-CFC generation; furthermore,
FLK1 cells produce fewer BL-CFCs
(Chung et al., 2002;
Faloon et al., 2000
;
Schuh et al., 1999
). Because
flow cytometry is a more rapid and convenient assay than BL-CFC production, we
employed FLK1 expression to screen for potential rescue of the
Arnt/ mesoderm defect using a battery of
growth factors. Factors including bFGF, VEGF, TGFß1, TGFß3, BMP2,
BMP4, ANG1, ANG2 and EPO were added, in multiple combinations, to
Arnt+/+ and Arnt/ EB
cultures, and FLK1 expression was analyzed by flow cytometry
(Table 1). Various growth
factor combinations containing BMPs, TGFßs, VEGF and/or bFGF resulted in
mild stimulation of FLK1+ cell numbers in 3.5-day
Arnt+/+ cultures, whereas hypoxia resulted in a marked
FLK1 increase (Table 1,
Experiment 1). One additional day of normoxic differentiation further
increased FLK1+ cells in Arnt+/+ EBs (4.5
days). However, FLK1+ cell numbers decreased on day 4.5 under
hypoxia, or in the presence of various growth factors
(Table 1). By contrast, no
treatment yielded a significant effect on FLK1+ cell numbers in two
independent Arnt/ cell lines
(Table 1, Experiment 1).
Experiments using EPO, ANG1 and/or ANG2 in combination with other growth
factors also had no effect on the FLK1+ cell population in
Arnt+/+ or Arnt/
cultures (Table 1, Experiments
2 and 3). Therefore, Arnt/ EBs appear to be
deficient in HIF target(s) that remain unidentified at this time.
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Discussion |
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In this report, we show that hypoxia influences the kinetics of early EB
differentiation. First, the expression of the mesodermal T-box gene
Bry was induced one day earlier under hypoxic conditions, as was
Bmp4, a gene encoding a growth factor involved in the ventralization
of early mesoderm and in blood formation
(Czyz and Wobus, 2001;
Hogan, 1996
;
Jones et al., 1996
;
Winnier et al., 1995
).
However, although BRY-GFP+ cell numbers are increased, the
induction is not as considerable as FLK1 expression. This could be interpreted
in two ways. First, flow cytometry is an underrepresentation of true
BRY+ cell numbers. Second, hypoxia increases the amount of
Bry transcript per cell but not the overall number of BRY+
cells. Significantly, Fehling et al. reported that BMP4 expression is limited
to the BRY+FLK1+ population of cells in EB cultures
(Fehling et al., 2003
),
supporting its role as a growth factor involved in mesodermal progenitors that
give rise to BL-CFCs. We determined that Bmp4 transcripts are
deficient in Arnt/ EBs, suggesting an
indirect role of HIF
/ARNT in mesoderm and hemangioblast development. In
the developing mouse embryo, mesoderm emerges from the primitive streak at
6.5-7.0 dpc. Of note, we have demonstrated that
Arnt/Arnt2/ embryos
die prior to 7.0 dpc, possibly because of inappropriate mesoderm
differentiation (Keith et al.,
2001
). Hypoxia also increased the expression of the Flk1
transcript, the number of FLK1+ cells and the number of BL-CFCs
generated. Moreover, hypoxia influenced the kinetics of BL-CFCs by
accelerating their interval of appearance, peaking about one day earlier in
hypoxic cultures compared with normoxic ones. Thus, it appears that low
O2 triggers an accelerated and increased commitment of mesoderm to
hemangioblast progenitors. These hypoxic responses are likely to promote the
O2-delivering capacity of the embryo in accord with increased
metabolic demand.
Previous analysis revealed that the frequency of BL-CFCs is lower than the
percentage of FLK1+ cells. We demonstrate that the kinetics of
BL-CFC production is shorter than of FLK1 expression, as FLK1+
cells are maintained beyond the narrow window of time during which BL-CFCs can
be generated. Thus, FLK1+ cells are a heterogeneous population,
only a fraction of which represents a true hemangioblast progenitor. FLK1
surface expression is maintained in more mature cell types, such as
endothelial cells. Nevertheless, FLK1 has been used as an experimental readout
for mesoderm commitment to hemangioblasts because its expression appears to be
necessary for BL-CFC formation (Faloon et
al., 2000). Thus, FLK1 surface expression is a useful tool in
screening cultures for BL-CFC potential, demonstrating that an assortment of
growth factors are not sufficient to rescue
Arnt/ ES cells.
The defect in Arnt/ BL-CFC formation
appears to be at the level of mesoderm commitment to hemangioblast cell fate
and/or subsequent hemangioblast differentiation, as
Arnt/ cells produce a preponderance of
transitional colonies at the apparent expense of hemangioblasts. A similar
phenotype was described for both cells and mice with a null mutation in the
gene encoding the bHLH transcription factor SCL. In vivo, SCL is essential for
hematopoiesis and vascular remodeling
(Robb et al., 1995;
Shivdasani et al., 1995
;
Visvader et al., 1998
); in
vitro, Scl/ EBs fail to generate BL-CFCs but
produce transitional colonies (Faloon et
al., 2000
; Robertson et al.,
2000
). Thus, both Arnt and Scl appear to
regulate hemangioblast development from early mesoderm precursors. Although
these results suggest that the hemangioblast defect should lead to a complete
absence of endothelial development, the fact that
Scl/ and
Arnt/ embryos are able to establish a
primary vascular system may be due to the existence of independent sources of
endothelial precursors, as have been shown in the avian system
(Pardanaud and Dieterlen-Lievre,
1999
). Unfortunately, we are unable to explore this possibility
further because it is difficult to assess somitic mesoderm in ES cell
cultures.
FLK1 is a cell-surface receptor for VEGF, and hemangioblast numbers are elevated in response to VEGF in methylcellulose assays. We have demonstrated that VEGF expression is stimulated under hypoxic conditions in wild-type EBs, and the number of BL-CFCs increases with the addition of VEGF to differentiating Arnt+/+ EBs. Although Vegf+/ and Vegf/ EBs produce a significant number of FLK1+ cells, they are still defective in generating appropriate BL-CFCs. Thus, it appears that appropriate levels of VEGF are required during the differentiation of EBs into hemangioblasts but not FLK1+ cells.
Although our data support a role for VEGF in the production of
hemangioblast colonies, other factors are essential. Faloon et al. suggest
that bFGF mediates hemangioblast proliferation while VEGF regulates blast
migration (Faloon et al.,
2000). Moreover, they were able to generate wild-type levels of
FLK1+ cell numbers from Scl/ ES
cells, and could further induce their numbers with the addition of bFGF during
EB differentiation. However, they failed to determine whether the
FLK1+ cells generated with the addition of bFGF were rescued for
their ability to generate BL-CFC colonies. Indeed, we stimulated
FLK1+ cell numbers with the addition of VEGF and/or bFGF in
Vegf mutant clones, but not in two independent
Arnt/ clones
(Fig. 5C). Again,
Arnt/ clones may be blocked at transitional
stages, which are refractory to growth factor signaling, as we have discovered
that multiple combinations of a large number of growth factors were
insufficient to rescue FLK1 expression. Importantly, in a definitive
co-culture experiment, we demonstrated that the
Arnt/ BL-CFC defect is rescued in the
presence of wild-type cells during EB differentiation, but FLK1+
cell numbers failed to be stimulated when the Arnt+/+ and
Arnt/ cells were separated by 3 µm
transwells. These results suggest that signaling via cell surface or poorly
diffusible molecules provided by the Arnt+/+ cells in
mixed EBs can induce proper differentiation of
Arnt/ ES cells into FLK1+ cells.
Further experiments will focus on the identification of such a factor(s).
In conclusion, we demonstrated that ARNT, as a subunit of HIF, is important
for the generation of the common precursors of cells (blood vessels and blood
cells) that supply O2 and nutrients to a growing embryo. Our model
suggests that in response to low O2, HIF first stimulates
Bry, a mesoderm gene, and BMP4, a mesodermal promoting factor. These
in vitro assays suggest that the hypoxic environment supports further mesoderm
maturation, such as the emergence of transitional colonies and their
subsequent differentiation into appropriate numbers of hemangioblasts (see
Fig. 7). Lack of ARNT and an
improper hypoxic response results in a block in differentiation, whereby
Arnt/ cultures are arrested and accumulate
at the transitional stage. Delayed differentiation and decreased numbers of
hemangioblast progenitors are likely to contribute to the vascular and
hematopoietic defects noted in the Arnt/ and
Hif1/ embryos. Therefore, development
of the blood and vascular systems is regulated at very early stages by
appropriate responses to O2 availability.
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ACKNOWLEDGMENTS |
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Footnotes |
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