1 Department of Pathology and Immunology, Washington University School of
Medicine, St. Louis, MO, USA
2 Department of Pediatrics and Center for Human Genetics and Molecular Pediatric
Disease, University of Rochester Medical Center, Rochester, NY, USA
Author for correspondence (e-mail:
kchoi{at}immunology.wustl.edu)
Accepted 3 September 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Hemangioblast, Hematopoiesis, Vasculogenesis, FLK1, SCL, Mouse
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gene-targeting studies have demonstrated that FLK1 (Kdr Mouse
Genome Informatics), a receptor tyrosine kinase, and SCL, a basic
helix-loop-helix transcription factor, are crucial for hematopoietic and
endothelial cell development. In mouse embryos, Flk1 expression can
be detected in the presumptive mesodermal yolk sac blood island progenitors as
early as E7 (Yamaguchi et al.,
1993; Dumont et al.,
1995
). Mice deficient in FLK1 do not develop blood vessels or yolk
sac blood islands, and die between E8.5 and E9.5
(Shalaby et al., 1995
). In
chimeric aggregation studies with wild-type embryos,
Flk1-/- ES cells fail to participate in vessel formation
or to contribute to primitive or definitive hematopoiesis, suggesting that
Flk1 inactivation results in cell autonomous endothelial and
hematopoietic defects (Shalaby et al.,
1997
). Mice carrying homozygous mutations at the Scl
locus die at around E10.5 because of defective embryonic hematopoiesis
(Shivdasani et al., 1995
;
Robb et al., 1995
). The
requirement of SCL in adult hematopoietic system has been shown in chimeric
mice generated by injecting Scl-/- ES cells into the
wild-type blastocysts (Porcher et al.,
1996
). None of the hematopoietic cells in these chimeric mice
developed from Scl-/- ES cells, suggesting a functional
role for SCL in adult hematopoiesis. Subsequent studies have shown that
vasculogenesis in the Scl-/- yolk sac occurs normally, but
that remodeling of the primary vascular plexus is defective
(Visvader et al., 1998
;
Elefanty et al., 1999
).
The in vitro differentiation model of ES cells has proven to be valuable
for studies of cell lineage development. Hematopoietic cells develop within
embryoid bodies (EBs, in vitro differentiated ES cells) faithfully following
in vivo developmental kinetics (Kennedy et
al., 1997; Choi et al.,
1998
; Palis et al.,
1999
). As in the developing embryo, primitive erythroid cells
develop prior to definitive hematopoietic populations
(Keller et al., 1993
;
Palis et al., 1999
).
Endothelial cells within EBs also follow similar kinetics, in that they
develop from FLK1+ mesodermal cells
(Vittet et al., 1996
;
Nishikawa et al., 1998
;
Nishikawa, 2001
). By using in
vitro differentiated ES cells, we previously identified blast colony forming
cells (BL-CFCs) as a long pursued common progenitor of hematopoietic and
endothelial cells, the hemangioblast (Choi
et al., 1998
) (reviewed by
Choi, 2002
). More importantly,
BL-CFCs are a transient cell population: they develop prior to primitive
erythroid cells, are most abundant in day 2.75-3.25 EBs and disappear shortly
thereafter.
We have previously demonstrated that BL-CFCs expressed FLK1
(Faloon et al., 2000) and that
Scl-/- EBs failed to generate blast colonies, the progeny
of BL-CFCs, in vitro (Faloon et al.,
2000
; Robertson et al.,
2000
). These studies suggest that SCL is crucial for hemangioblast
development and that the hemangioblast can be identified as the
FLK1+SCL+ cell population. To understand better the
relationship between FLK1 and SCL expression in the differentiation of
hematopoietic and endothelial cell lineages, we have introduced a
non-functional human CD4 gene (CD4) encoding extracellular and
transmembrane domains into one allele of Scl. This strategy allows us
to detect SCL-expressing cells by using monoclonal antibodies against human
CD4 and flow cytometry analyses. Kinetic analyses of FLK1 and human CD4
expression of in vitro differentiated Scl+/CD4 ES cells
and cell sorting experiments for hemangioblast, hematopoietic and endothelial
cells demonstrated that hematopoietic and endothelial cells developed via the
sequential generation of FLK1- and SCL- expressing cells.
In this paper, CD4 refers to the non-functional human CD4 gene/protein.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ES differentiation, blast colony and hematopoietic replating
Mouse ES cells were maintained on STO feeder cells in the presence of
leukemia inhibitory factor (LIF). EBs were generated as described
(Kennedy et al., 1997;
Choi et al., 1998
). Blast
colonies were generated by replating sorted CD4+ cells from day
2.75-3 EBs in the presence of VEGF (5 ng/ml), kit ligand (KL, 1% conditioned
medium or 100ng/ml purified) and D4T endothelial cell conditioned medium (CM)
(Kennedy et al., 1997
;
Choi et al., 1998
) at 25%.
Erythroid and myeloid colony assays were carried out as described previously
(Faloon et al., 2000
). Briefly,
cells sorted from day 6 and day 8 EBs were cultured in methyl cellulose
containing 10% plasma-derived serum (PDS, Antech; Texas), 5% protein free
hybridoma medium (PFHM2, Gibco/BRL), ascorbic acid (12.5 µg/ml),
L-glutamine (2 mM), transferrin (300 µg/ml; Boehringer Mannhein) and MTG
(4.5x10-4 M), together with the following cytokines: KL (1%
conditioned medium), IL3 (1% conditioned medium), Epo (2 units/ml), IL1 (5
ng/ml), IL6 (5 ng/ml), IL11 (10 ng/ml), G-CSF (30 ng/ml), M-CSF (5 ng/ml) and
GM-CSF (3 ng/ml). Hematopoietic colonies were counted 7-10 days later. IL1,
IL6, IL11, G-CSF and M-CSF were purchased from R&D Systems. KL was
obtained from a medium conditioned by CHO cells transfected with a KL
expression vector (kindly provided by Genetics Institute). Epo was purchased
from Amgen (Thousand Oaks, CA), and IL3 was obtained from a medium conditioned
by X63 Ag8-653 myeloma cells transfected with a vector expressing IL3
(Karasuyama and Melchers,
1988
).
FACS analysis
For FACS analysis of FLK1 and CD4 expression, EBs were treated with 7.5 mM
EDTA/PBS (pH 7.4) for 2 minutes. Cells were centrifuged, resuspended in
staining medium (4% FCS in PBS), passed through a 20-gauge needle to generate
single cell suspension, and counted. After centrifugation, cells were
resuspended to a density of 5x106 cells/ml in 2.4G2
supernatant to block antibodies from binding to Fc receptors II and III (CD16
and CD32) (Unkeless, 1979).
Cells were placed into each well of a V-shaped 96-well plate at
5x105 cells/well followed by incubation on ice for 30
minutes. Subsequently, biotinylated mouse anti-human CD4 monoclonal antibody
(CALTAG), freshly diluted in wash buffer (4% FCS in PBS), was added into each
well and incubated on ice for 15 minutes. After three washes, freshly diluted
streptavidin-allophycocyanin (Sav-APC; Pharmingen) and phycoerythrin
(PE)-conjugated anti-FLK1 monoclonal antibody (Pharmingen) were added and
incubated on ice in the dark for 15 minutes. Cells were washed three times,
re-suspended in wash buffer, and transferred to 5 ml polypropylene tubes for
analysis. A three-color FACS analysis of FLK1, human CD4 and endothelial or
hematopoietic markers was carried out by staining cells first with
endothelial/hematopoietic markers. FITC-conjugated anti-mouse CD31,
FITC-conjugated anti-mouse CD34 or FITC-conjugated anti-mouse CD45 was added
directly. When cells were stained with non-labeled anti-mouse VE-cadherin and
anti-mouse Ter-119 antibodies, FITC-conjugated goat anti-rat IgG and
FITC-conjugated goat anti-rat IgG2b, respectively, were used to
amplify the signals. All the antibodies were purchased from Pharmingen. Cells
were subsequently stained with anti-human CD4 and anti-FLK1 antibodies as
described above. Cells were analyzed on a FACS Caliber (Becton-Dickinson).
FACS data were analyzed with CellQuest software (Becton-Dickinson).
Cell sorting and in vitro cultures of sorted cell populations
For FACS-cell sorting, single cell suspensions were prepared the same way
as the FACS analyses, except that the EB cells were dissociated with trypsin
(0.08%)/EDTA (0.36 mM)/PBS instead of 7.5 mM EDTA/PBS. Double-color staining
and sorting for FLK1 and human CD4 cells were performed the same way as for
FACS analysis. Prior to sorting, stained cells were filtered through 40-µm
nylon mesh. Cells were sorted using FACS MoFlo (Becton-Dickinson), and the
sorted cells were reanalyzed on a FACS Caliber.
FLK1+CD4- or FLK1+CD4+ sorted
cells were cultured for an additional 20-48 hours in a bacterial petri dish in
IMDM media containing 15% pre-selected FCS, ascorbic acid (50 µg/ml),
L-glutamine (2 mM) and MTG (4.5x10-4 M) at a cell density of
2x105/ml.
Endothelial cell cultures and immunohistochemical staining
Cells sorted from day 6 EBs were plated onto type IV collagen
(Sigma)-coated, 24-well plates in IMDM media containing 15% preselected FCS,
ascorbic acid (50 µg/ml), L-glutamine (2 mM), MTG
(4.5x10-4 M) and VEGF (50 ng/ml) at a cell density of
2x104/well. Cells were cultured in humidified 37°C
incubator with 5% CO2 for 3-4 days. For immunohistochemical
staining, adherent cells were washed with PBS, fixed for 10 minutes in PBS
containing 4% paraformaldehyde (PFA) at 4°C, and washed twice (10 minutes
each) in PBS. Following the wash, the endogenous peroxidase was quenched in
methanol/30% hydrogen peroxide/10% sodium azide (50:10:1) for 1 hour at
4°C. Cells were washed twice and blocked with PBS containing 1% goat
serum, 0.2% bovine serum albumin and 2% skim milk for 1 hour. Cells were then
incubated with biotinylated anti-mouse CD31 (Pharmingen) overnight at 4°C.
After three washes, cells were incubated with streptavidin-horseradish
peroxidase (Pharmingen) for 1 hour at room temperature. Cells were then washed
three times and incubated with a DAB kit to develop the color (Vector).
In situ hybridization
Sorted CD4+ and CD4- cells, from day 5 EBs, were
fixed in freshly prepared 4% paraformaldehyde in PBS. Cells were dehydrated
through graded ethanols, xylene and embedded in paraffin wax. Sections (4
µm) were adhered to Superfrost Plus (VWR) microscope slides. Sections of
E8.5 mouse embryos were placed on the slides and served as positive controls
for Scl expression (Silver and
Palis, 1997). In situ hybridization was performed essentially as
described (McGrath et al.,
1999
), except probes were synthesized at 4.2x109
dpm/µg, and hybridization occurred at 50°C. Replicate slides were
probed with a sense control probe and no signal above background was detected
(not shown). Cells were photographed in brightfield and darkfield with a SPOT
RT slider (Diagnostic Instruments) digital camera. Images were processed,
pseudocolored and merged using Photoshop (Adobe Systems) and Fovea Pro 2
(Reindeer Graphics).
Gene expression analysis
RNA was purified from sorted FLK1+CD4+,
FLK1+CD4-, and FLK1-CD4- cells
(from day 2.75 EBs), reverse transcribed, and poly-A tailed using terminal
transferase. Total cDNA was amplified using oligo dT as a primer
(5'GTTAACTCGAGAATTCT243')
(Brady et al., 1990). After one
round of re-amplification using 1µl of the primary PCR products as
template, PCR products were separated by agarose gel electrophoresis,
transferred to a nylon membrane, and hybridized with 32P randomly
primed cDNA probe corresponding to the 3' end of the L32, Flk1, Scl,
Gata1, Gata2 and Lmo2 genes. After the hybridization, the blot
was washed at high stringency and exposed to an X-ray film.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Kinetics of the development of FLK1 and CD4-expressing cells during
EB differentiation
To establish that CD4 expression truly correlates with that of
Scl, we first sorted CD4+ and CD4- cells and
subjected them to in situ hybridization for Scl expression. As shown
in Fig. 2, the sorted
CD4+ cells showed strong hybridization to Scl antisense
probe (Fig. 2A-D), while
CD4- cells were negative for Scl expression
(Fig. 2E-H). The levels of
Scl expression in the sorted CD4+ cells are similar to the
endogenous levels in blood island cells of the E8.5 mouse embryo
(Fig. 2I). These studies
indicate that CD4 expression can be used as a surrogate marker for
Scl expression.
|
After CD4+ cells were shown to represent SCL expressing cells
accurately, kinetic analyses of FLK1 and CD4 expression were performed using
in vitro differentiated Scl+/CD4 ES cells.
Undifferentiated ES cells do not express FLK1 or SCL
(Choi et al., 1998;
Faloon et al., 2000
). Upon
differentiation, cells expressing FLK1 developed first in EBs and
CD4-expressing cells were detected from day 2.75 in developing EBs
(Fig. 2J), although the levels
of CD4 expression at this stage were low. The low CD4 expression in early EBs
could reflect the weak Scl promoter activity in early development and
is consistent with studies by Elefanty, who characterized knock-in mice
carrying a lacZ gene at the Scl locus
(Elefanty et al., 1998
;
Elefanty et al., 1999
). Cells
expressing CD4 increased significantly over the next 24-48 hours
(Fig. 2J). About 40% of the
total EB cells expressed CD4 by day 5, and
75% of the total EB cells
expressed CD4 by day 6. The percentage of CD4+ cells decreased
thereafter and reached to
20% by day 8. At earlier time points (days
2.75-3), all the CD4+ cells also expressed FLK1. EBs from days 4-6
contained three distinct cell populations as follows: cells expressing only
FLK1 (FLK1+CD4-), cells expressing only CD4
(FLK1-CD4+), and cells expressing both FLK1 and CD4
(FLK1+CD4+). In later EBs (day 8), the number cells
expressing both FLK1 and CD4 was significantly lower, and there were
predominantly FLK1+CD4- or
FLK1-CD4+ cells.
Developmental relationship between FLK1+CD4-,
FLK1+CD4+ and FLK1-CD4+ cell
populations
FACS analysis for FLK1 and CD4 expression suggested that FLK1+
cells developed first followed by CD4+ cells. To understand better
the developmental relationship between FLK1+CD4-,
FLK1+CD4+ and FLK1-CD4+ cells, we
first FACS-sorted FLK1+ and FLK1- cells from day 2.5 EBs
and then cultured them separately for an additional 20 hours. FLK1+
cells progressed to give rise to FLK1+CD4+ cells after
10 hours of in vitro culture, and FLK1-CD4+ cells were
readily detected after 20 hours of culture. FLK1- cells progressed
first to FLK1+ and then to FLK1+CD4+ cells
with time (Fig. 3A). We
subsequently FACS-sorted FLK1+CD4+ and
FLK1+CD4- cell populations from day 4 EBs, cultured them
for an additional 20 hours, and analyzed them for FLK1 and CD4 expression. As
shown in Fig. 3B,
FLK1+CD4+ cells readily gave rise to
FLK1-CD4+ cells, indicating that FLK1 is downregulated
within the FLK1+CD4+ cells to generate
FLK1-CD4+ cell populations. Consistently,
FLK1+CD4- cells first gave rise to
FLK1+CD4+ and then to FLK1-CD4+
cells upon an additional 20-hour culture. FLK1-CD4+
cells further increased after 48 hours of culture (not shown). These studies
clearly argue that there is a distinct, developmental succession between
FLK1+CD4-, FLK1+CD4+ and
FLK1-CD4+ cells. The FLK1+CD4-
cell population will initially develop. The Scl gene will be turned
on within the FLK1+CD4- cells to give rise to the
FLK1+CD4+ cell population. Subsequently, the
Flk1 gene will be downregulated within the
FLK1+CD4+ cells to finally give rise to the
FLK1-CD4+ cells.
|
FLK1+SCL+ cells from day 2.75 EBs are enriched
for the hemangioblast
As discussed earlier, BL-CFCs express FLK1
(Faloon et al., 2000) and
Scl-/- EBs fail to generate blast colonies that are the
progeny of BL-CFCs (Faloon et al.,
2000
; Robertson et al.,
2000
). To determine if the BL-CFC cell population could be
identified as FLK1+SCL+, day 2.75 EB cells were
subjected to FACS analysis and cell sorting. If FLK1 and SCL are true
determinants of the hemangioblast, it was expected that
FLK1+CD4+ cells were enriched for BL-CFCs. Therefore,
FLK1+CD4+, FLK1+CD4- and
FLK1-CD4- cells from day 2.75 EBs were sorted and
subjected to blast colony assays. As shown in
Fig. 4A, blast colonies
developed predominantly from sorted FLK1+CD4+ cells, not
FLK1+CD4- or FLK1-CD4- cells. The
small number of blast colonies that developed from
FLK1+CD4- cells is most likely to be due to the
CD4low cells that could have been sorted as a negative cell
population. Furthermore, secondary EBs mainly developed from
FLK1-CD4- cells, strongly supporting the idea that
FLK1-CD4- cells still contained undifferentiated ES
cells.
|
To characterize further the FLK1+CD4+,
FLK1+CD4- and FLK1-CD4- cells
present within day 2.75 EBs, they were also subjected to gene expression
analyses. RNA from FLK1+CD4+,
FLK1+CD4- and FLK1-CD4- cells was
subjected to global amplification of mRNA transcripts
(Brady et al., 1990). The
amplified PCR products were analyzed for the expression of Flk1, Scl,
Gata1, Gata2 and Lmo2 (Fig.
4B). As expected, Flk1 was expressed within
FLK1+CD4- and FLK1+CD4+ cells.
Scl, Gata1, Gata2, and Lmo2 expression was greatly raised
within FLK1+CD4+ cells compared with
FLK1+CD4- cells. None of these genes was expressed in
FLK1-CD4- cells. Taken together, our results strongly
support the notion that hemangioblasts can be identified as
FLK1+SCL+ cells.
FLK1 and SCL expression in hematopoietic and endothelial cells
To determine if FLK1 and SCL expression can define hematopoietic and
endothelial cell populations, the nature of FLK1+CD4-,
FLK1-CD4+, and FLK1+CD4+ cell
populations, present in later stages of EB development (days 5-6), was
determined by three-color FACS analyses for FLK1, CD4 and hematopoietic or
endothelial cell markers. Cells gated on VE-cadherin+,
CD31+ (PECAM-1+), CD34+, Ter-119+,
or CD45+ were analyzed for FLK1 and CD4 expression. As shown in
Fig. 5, cells expressing
VE-cadherin, an endothelial cell marker, expressed both FLK1 and CD4, while
cells expressing Ter119, an erythroid marker, expressed CD4 but not FLK1. CD31
and CD34, markers of both hematopoietic and endothelial cells, were expressed
in both FLK1+CD4+ and FLK1- CD4+
cells. CD45, a marker normally used for hematopoietic cells, was expressed in
both FLK1+CD4+ and FLK1-CD4+ cells
at day 6. CD45 was predominantly expressed in FLK1-CD4+
cells from later stages of EBs (day 8, not shown).
|
To ascertain if hematopoietic and endothelial progenitors can be isolated based on SCL and FLK1 expression, we FACS-sorted FLK1+CD4-, FLK1+CD4+ and FLK1-CD4+ cells from day 6 EBs, and subjected them to hematopoietic and endothelial cell assays. For the hematopoietic progenitor studies, sorted cells were replated in methylcellulose cultures with hematopoietic factors. As shown in Fig. 6A, hematopoietic colonies developed from both FLK1+CD4+ and FLK1-CD4+ cells. FLK1-CD4+ cells predominantly gave rise to erythroid colonies, while FLK1+CD4+ cells gave rise to macrophage and bi-potential erythroid/macrophage colonies. The endothelial progenitors were assayed by replating sorted cells onto type IV collagen-coated plates with VEGF and cultured for 4 days. Afterwards, the adherent cells were stained for CD31. As shown in Fig. 6B, endothelial cells developed from FLK1+CD4- and FLK1+CD4+, but not from FLK1-CD4- cells. No adherent cells developed from FLK1-CD4+ cells.
|
FLK1+CD4+ cells were rarely present in day 8 EBs, and two distinct cell populations, FLK1+CD4- and FLK1-CD4+, were readily observed (Fig. 2). Again, these two cell populations were sorted and examined for their potential to generate hematopoietic or endothelial cells in cultures. As shown in Fig. 6C, all the hematopoietic colonies developed from FLK1-CD4+ cells, while the endothelial cells still developed from FLK1+CD4- cells (not shown). Taken together, we conclude that hematopoietic progenitors initially develop from FLK1+CD4+ and FLK1-CD4+ cells and then from FLK1-CD4+ cells in later stages of EBs.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The analyses of sorted cell populations have demonstrated that
FLK1+ cells isolated from day 2.5 EBs progressed to give rise
initially to FLK1+CD4+ cells and subsequently to
FLK1-CD4+ cells. Consistently, sorted
FLK1+CD4- cells from day 4 EBs progressed to give rise
to FLK1+CD4+ and then to FLK1-CD4+
cell populations when cultured for an additional 24-48 hours in vitro, while
FLK1+CD4+ cells proceeded to give rise to
FLK1-CD4+ cells. As the hematopoietic progenitors were
present within FLK1-CD4+ and
FLK1+CD4+ cells in early EBs (day 4-6) and in
FLK1-CD4+ cells in later EBs (day 8,
Fig. 2), we conclude that FLK1
expression within hematopoietic progenitors is downregulated
(Fig. 7). Consistent with this
interpretation, there were only FLK1-CD4+ or
FLK1+CD4- cell populations present in later stages of
EBs (day 8). Previous studies have also demonstrated that hematopoietic
progenitors were enriched within the FLK1+ cell populations derived
from early EBs, but not later stages of EBs
(Kabrun et al., 1997).
Similarly, the FLK1+ cell population from E8.5 yolk sacs and whole
embryos contained hematopoietic progenitors, while few FLK1+ cells
present in day 12 fetal livers contained hematopoietic potential
(Kabrun et al., 1997
).
Elefanty and colleagues knocked-in a bacterial lacZ gene to the
Scl locus to follow SCL-expressing cells
(Elefanty et al., 1998
;
Elefanty et al., 1999
).
Histochemical staining of Scl+/lacZ embryos for
ß-galactosidase activity showed that lacZ was expressed in
hematopoietic and endothelial cells, as well as in the developing brain. Cell
sorting and replating studies of ß-galactosidase+ cells from
fetal livers showed that erythroid and myeloid progenitors were present within
ß-galactosidase+ cells. Furthermore,
ß-galactosidase+ cell fractions from the bone marrow were
enriched for erythroid, myeloid, lymphoid and CFU-S12 progenitors. These
studies support the notion that SCL is expressed in hematopoietic progenitors,
which could also include hematopoietic stem cells.
|
Our studies have also demonstrated that the development of endothelial
cells can be followed by FLK1 and SCL expression
(Fig. 7). Replating studies
have demonstrated that endothelial cells developed from two distinct cell
populations, FLK1+CD4- and
FLK1+CD4+ cells. The nature of the endothelial cells
developing from the FLK1+CD4- and
FLK1+CD4+ cells is currently not known. Given the
findings that vascular development occurs normally in
Scl-/- embryos, but that subsequent vascular remodeling is
defective in these embryos (Visvader et
al., 1998), it is possible that FLK1+CD4+
derived endothelial cells represent terminally differentiated mature
endothelial cells. In avian systems, it has been well demonstrated that
different mesodermal regions produce endothelial progenitors
(Pardanaud et al., 1996
). The
somatopleural mesoderm, adjacent to the ectoderm, will give rise only to
endothelial cell populations. The splanchnopleural mesoderm, adjacent to the
endoderm, will give rise to both hematopoietic and endothelial cells. Given
these observations, it is possible that endothelial cells that develop closely
together with hematopoietic cells will express Scl, while endothelial
cells that do not associate with hematopoietic cells will not express
Scl. Further studies are required to address these issues.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bedinger, P., Moriarty, A., von Borstel, R. C., 2nd, Donovan, N. J., Steimer, K. S. and Littman, D. R. (1988). Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4. Nature 334,162 -165.[CrossRef][Medline]
Brady, G., Barbara, M. and Iscove, N. (1990). Representative in vitro cDNA amplification from individual hematopoietic cells and colonies. Meth. Mol. Cell. Biol. 2, 17-25.
Choi, K. (2002). The hemangioblast: A common progenitor of hematopoietic and endothelial cells. J. Hematotherapy Stem Cell Res. 11,91 -101.[CrossRef][Medline]
Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, J. and
Keller, G. (1998). A common precursor for hematopoietic and
endothelial cells. Development
125,725
-732.
Drake, C. J. and Fleming, P. A. (2000).
Vasculogenesis in the day 6.5 to 9.5 mouse embryo.
Blood 95,1671
-1679.
Dumont, D. J., Fong, G. H., Puri, M. C., Gradwohl, G., Alitalo, K. and Breitman, M. L. (1995). Vascularization of the mouse embryo: a study of flk-1, tek, tie, and vascular endothelial growth factor expression during development. Dev. Dyn 203, 80-92.[Medline]
Elefanty, A. G., Begley, C. G., Metcalf, D., Barnett, L.,
Kontgen, F. and Robb, L. (1998). Characterization of
hematopoietic progenitor cells that express the transcription factor SCL,
using a lacZ "knock-in" strategy. Proc. Natl. Acad.
Sci. USA 95,11897
-11902.
Elefanty, A. G., Begley, C. G., Hartley, L., Papaevangeliou, B.
and Robb, L. (1999). SCL expression in the mouse embryo
detected with a targeted lacZ reporter gene demonstrates its localization to
hematopoietic, vascular, and neural tissues. Blood
94,3754
-3763.
Faloon, P., Arentson, E., Kazarov, A., Deng, C. X., Porcher, C.,
Orkin, S. and Choi, K. (2000). Basic fibroblast growth factor
positively regulates hematopoietic development.
Development 127,1931
-1941.
Gering, M., Rodaway, A. R. F., Gottgens, B., Patient, R. K. and
Green, A. R. (1998). The SCL gene specifies haemangioblast
development from early mesoderm. EMBO J.
17,4029
-4045.
Kabrun, N., Buhring, H. J., Choi, K., Ullrich, A., Risau, W. and
Keller, G. (1997). Flk-1 expression defines a population of
early embryonic hematopoietic precursors. Development
124,2039
-2048.
Karasuyama, H. and Melchers, F. (1988). Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur. J. Immunol. 18, 97-104.[Medline]
Keller, G., Kennedy, M., Papayannopoulou, T. and Wiles, M. V. (1993). Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol. Cell. Biol. 13,473 -486.[Abstract]
Kennedy, M., Firpo, M., Choi, K., Wall, C., Robertson, S., Kabrun, N. and Keller, G. (1997). A common precursor for primitive erythropoiesis and definitive haematopoiesis, Nature 386,488 -493.[CrossRef][Medline]
Liao, E. C., Paw, B. H., Oates, A. C., Pratt, S. J.,
Postlethwait, J. H. and Zon, L. I. (1998). SCL/Tal-1
transcription factor acts downstream of cloche to specify hematopoietic and
vascular progenitors in zebrafish, Genes Dev.
12,621
-626.
McGrath, K. E., Koniski, A. D., Maltby, K. M., McGann, J. and Palis, J. (1999). Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4. Dev. Biol. 213,442 -456.[CrossRef][Medline]
Murray, P. (1932). The development `in vitro' of blood of the early chick embryo. Strangeways Res. Labor. Cambridge 497-521.
Nishikawa, S. I. (2001). A complex linkage in the developmental pathway of endothelial and hematopoietic cells. Curr. Opin. Cell. Biol. 13,673 -678.[CrossRef][Medline]
Nishikawa, S. I., Nishikawa, S., Hirashima, M., Matsuyoshi, N.
and Kodama, H. (1998). Progressive lineage analysis by cell
sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of
endothelial and hemopoietic lineages. Development
125,1747
-1757.
Palis, J., Robertson, S., Kennedy, M., Wall, C. and Keller,
G. (1999). Development of erythroid and myeloid progenitors
in the yolk sac and embryo proper of the mouse.
Development 126,5073
-5084.
Pardanaud, L., Luton, D., Prigent, M., Bourcheix, L. M., Catala,
M. and Dieterlen-Lievre, F. (1996). Two distinct endothelial
lineages in ontogeny, one of them related to hemopoiesis.
Development 122,1363
-1371.
Porcher, C., Swat, W., Rockwell, K., Fujiwara, Y., Alt, F. W. and Orkin, S. H. (1996). The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 86,47 -57.[Medline]
Robb, L., Lyons, I., Li, R., Hartley, L., Kontgen, F., Harvey, R. P., Metcalf, D. and Begley, C. G. (1995). Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. Proc. Natl. Acad. Sci. USA 92,7075 -7079.[Abstract]
Robertson, S. M., Kennedy, M., Shannon, J. M. and Keller, G.
(2000). A transitional stage in the commitment of mesoderm to
hematopoiesis requiring the transcription factor SCL/tal-1.
Development 127,2447
-2459.
Sabin, F. R. (1920). Studies on the origin of blood vessels and of red corpuscles as seen in the living blastoderm of the chick during the second day of incubation. Embryology 9, 213-262.
Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertsenstein, M., Wu, X. F., Breitman, M. L. and Schuh, A. C. (1995). Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376,62 -66.[CrossRef][Medline]
Shalaby, F., Ho, J., Stanford, W. L., Fischer, K. D., Schuh, A. C., Schwartz, L., Bernstein, A. and Rossant, J. F. (1997). A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell 89,981 -990.[Medline]
Shivdasani, R. A., Mayer, E. L. and Orkin, S. H. (1995). Absence of blood formation in mice the T-cell leukaemia oncoprotein tal-1/SCL. Nature 373,432 -434.[CrossRef][Medline]
Silver, L. and Palis, J. (1997). Initiation of
murine embryonic erythropoiesis: a spatial analysis.
Blood 89,1154
-1164.
Stainier, D. Y., Weinstein, B. M., Detrich, H. W.,
3rd, Zon, L. I. and Fishman, M. C. (1995). Cloche,
an early acting zebrafish gene, is required by both the endothelial and
hematopoietic lineages. Development
121,3141
-3150.
Unkeless, J. C. (1979). Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150,580 -596.[Abstract]
Visvader, J. E., Fujiwara, Y. and Orkin, S. H.
(1998). Unsuspected role for the T-cell leukemia protein
SCL/tal-1 in vascular development. Genes Dev.
12,473
-479.
Vittet, D., Prandini, M. H., Berthier, R., Schweitzer, A.,
Martin-Sisteron, H., Uzan, G. and Dejana, E. (1996).
Embryonic stem cells differentiate in vitro to endothelial cells through
successive maturation steps. Blood
88,3424
-3431.
Wagner, R. C. (1980). Endothelial cell embryology and growth. Adv. Microcirc. 9, 45-75.
Yamaguchi, T. P., Dumont, D. J., Conlon, R. A., Breitman, M. L.
and Rossant, J. (1993). flk-1, an flt-related receptor
tyrosine kinase is an early marker for endothelial cell precursors.
Development 118,489
-498.