HEMATOPOIESIS
Hemogenic and nonhemogenic endothelium can be distinguished by the activity
of fetal liver kinase (Flk)–1 promoter/enhancer during mouse embryogenesis
Hideyo Hirai, Minetaro Ogawa, Norio Suzuki, Masayuki Yamamoto, Georg Breier, Osam Mazda, Jiro Imanishi, and Shin-Ichi Nishikawa
Accumulating evidence in various species has suggested that the origin of
definitive hematopoiesis is associated
with a special subset of endothelial cells
(ECs) that maintain the potential to give
rise to hematopoietic cells (HPCs). In this
study, we demonstrated that a combination of 5ⴕ-flanking region and 3ⴕ portion of
the first intron of the Flk-1 gene (Flk-1 p/e)
that has been implicated in endotheliumspecific gene expression distinguishes
prospectively the EC that has lost hemogenic activity. We assessed the activity of
this Flk-1 p/e by embryonic stem (ES) cell
differentiation culture and transgenic mice
by using the GFP gene conjugated to this
unit. The expression of GFP differed from
that of the endogenous Flk-1 gene in that
it is active in undifferentiated ES cells and
inactive in Flk-1ⴙ lateral mesoderm. Flk-1
p/e becomes active after generation of
vascular endothelial (VE)–cadherinⴙ ECs.
Emergence of GFPⴚ ECs preceded that of
GFPⴙ ECs, and, finally, most ECs expressed GFP both in vitro and in vivo. Cell
sorting experiments demonstrated that
only GFPⴚ ECs could give rise to HPCs
and preferentially expressed Runx1 and
c-Myb genes that are required for the
definitive hematopoiesis. Integration of
both GFPⴙ and GFPⴚ ECs was observed
in the dorsal aorta, but cell clusters appeared associated only to GFPⴚ ECs.
These results indicate that activation of
Flk-1 p/e is associated with a process that
excludes HPC potential from the EC differentiation pathway and will be useful for
investigating molecular mechanisms underlying the divergence of endothelial
and hematopoietic lineages. (Blood. 2003;
101:886-893)
© 2003 by The American Society of Hematology
Introduction
During early embryogenesis, hematopoietic cells (HPCs) are
generated in close association with the development of the vascular
system. In the blood islands of the yolk sac where the earliest
hematopoietic cells appear, both hematopoietic and endothelial cell
(EC) lineages arise almost simultaneously from extraembryonic
mesoderm, thereby forming structures in which primitive erythrocytes are surrounded by a layer of angioblasts. These histologic
observations have led to the hypothesis that the 2 lineages arise
from a common precursor, the hemangioblasts.1 This concept is
supported by the shared expressions of a number of different genes
by both lineages.2-5 Following the process known as primitive
hematopoiesis, the major hematopoietic site shifts to the fetal liver
at midgestation and finally to the bone marrow. Before colonizing
fetal liver, the definitive type hematopoietic progenitors were
thought to be generated in a restricted region within the embryo.6-8
About the cellular origin of definitive hematopoiesis, several
possibilities have been proposed.9 An intraembryonic origin for
definitive hematopoiesis is supported by histologic observations
that clusters of hematopoietic cells are attached to the luminal
wall of the dorsal aorta, as if budding from the endothelial
cells.10 As the presence of such intra-aortic clusters has been
observed over many vertebrate species and correlates well with
development of the definitive hematopoietic cells,11-14 it was
proposed that at least a certain portion of definitive hematopoiesis derives from “the hemogenic endothelium.”15 The concept
of hemogenic endothelium is also supported by functional
studies investigating the potential of ECs to give rise to HPCs.
In avian systems, clonogenic analyses have demonstrated that
multipotent hematopoietic progenitors are generated only from
the aortic region.16 Jaffredo et al17 showed that hematopoietic
cells are derived from endothelial cells that had been labeled by
low-density lipoproteins injected into the circulation of chick
embryos, indicating that endothelial cells of the dorsal aorta can
function as hematopoietic progenitors. Moreover, we have
demonstrated that vascular endothelial (VE)–cadherin⫹ cells
that were purified from murine embryos can give rise to
hematopoietic cells.18
It is of importance to know biologic significance and mechanisms of the emergence of HPCs from a subset of ECs during
embryogenesis. We have established a culture system in which
embryonic stem (ES) cells differentiate into HPCs and ECs through
the proximal lateral mesoderm.19-21 Flk-1⫹ VE cadherin⫺ cells that
represent lateral mesodermal cells were induced first from ES cells.
The divergence of primitive type HPCs and Flk-1⫹ VE-cadherin⫹
ECs was shown to occur during the mesodermal stage.22 The
definitive type HPCs eventually diverged from the Flk-1⫹
From the Department of Microbiology, Kyoto Prefectural University of Medicine,
Kyoto, Japan; Department of Molecular Genetics, Faculty of Medicine, Kyoto
University, Kyoto, Japan; Center for Tsukuba Advanced Research Alliance and
Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan;
Max-Planck-Institute for Physiological and Clinical Research, Bad Nauheim,
Germany.
and Technology of Japan (nos. 12770506, 12670301, and 07CE2005), the Cell
Science Research Foundation, and Japanese Society for the Promotion of
Science “Research of Future” program.
Submitted February 28, 2002; accepted September 9, 2002. Prepublished
online as Blood First Edition Paper, September 19, 2002; DOI 10.1182/blood2002-02-0655.
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.
Supported by grants from the Ministry of Education, Culture, Sports, Science
© 2003 by The American Society of Hematology
886
Reprints: Hideyo Hirai, Department of Microbiology, Kyoto Prefectural
University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan; e-mail:
[email protected].
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BLOOD, 1 FEBRUARY 2003 䡠 VOLUME 101, NUMBER 3
VE-cadherin⫹ ECs.22 This in vitro culture system enables us to
dissect the differentiation process of HPCs and ECs from mesoderm. For further investigation of molecular mechanisms underlying the divergence of EC and HPC lineages, it is useful to drive the
genes of interest in a specific lineage at specific stages.
Recently, cis-acting regulatory elements of the murine fetal
liver kinase-1 (Flk-1) were studied by Kappel et al23 who showed
that a combination of a 5⬘-flanking region and 3⬘ portion of the first
intron (Flk-1 promoter/enhancer) was sufficient to direct ECspecific gene expression in vivo, although activity of the cis
element in Flk-1⫹ mesodermal cell was not known. We analyzed
the activity of this promoter/enhancer in ES cell differentiation in
vitro and unexpectedly found that these Flk-1 regulatory elements
are active only after commitment to ECs but not in lateral
mesodermal stage. A more interesting finding is that only ECs
negative for green fluorescence protein (GFP) driven by the Flk-1
promoter/enhancer (p/e) could give rise to HPCs. In this study, we
will show both in vitro and in vivo that Flk-1 p/e is a useful marker
for monitoring the EC maturation, which is inversely correlated
with the hemogenic potential.
HEMOGENIC AND NONHEMOGENIC ENDOTHELIUM
887
Figure 1. Outlines of experimental procedure. (A) Partial structure of the murine
Flk-1 locus and a reporter gene construct. The GFP gene is flanked by a Flk-1
promoter fragment (prom) spanning bp ⫺640 to bp ⫹299 and an enhancer sequence
between bp ⫹1677 and bp ⫹3947 in the first intron. (B) For induction of differentiation
in vitro, undifferentiated ES cells were transferred to a type IV collagen–coated plate
and incubated for 4 days in the absence of LIF. Cultured cells were harvested and
analyzed for expression of GFP and Flk-1 by flow cytometry. The Flk-1⫹ cells were
sorted for secondary culture on OP9 stromal cells. These cells were recovered after 1
to 5 days and analyzed for expression of GFP, VE-cadherin, CD45, and TER119 or for
hemangiogenic ability.
Materials and methods
Monoclonal antibodies (MoAbs), cell staining, and sorting
The MoAbs AVAS12 (anti–Flk-1)24 and VECD1 (anti–VE-cadherin)25 were
purified from hybridoma culture supernatants by protein G-Sepharose
columns (Pharmacia, Uppsala, Sweden) and labeled with allophycocyanin
(APC) by standard methods. The phycoerythrin (PE)–conjugated MoAbs,
anti-CD45, Ter119 (erythroid lineage marker), and anti-CD31 (platelet
endothelial cell adhesion molecule-1 [PECAM-1]) were purchased from
PharMingen (San Diego, CA).
Cells were blocked with normal mouse serum and labeled with
combinations of the above MoAbs. Stained cells were resuspended in Hank
balanced salt solution (GIBCO BRL) containing 1% bovine serum albumin
(Sigma, St Louis, MO) and 5 ␮g/mL propidium iodide (PI; Sigma) to
exclude dead cells. Cells were analyzed and sorted by fluorescenceactivated cell sorter (FACS) Vantage (Becton Dickinson Immunocytometry
Systems, San Jose, CA). Data were analyzed using the software CellQuest
(Becton Dickinson Immunocytometry Systems).
Plasmids
The pGLacZ-Flk-1p/e contains the ␤-galactosidase (LacZ) gene sequence
under the control of the ⫺640 bp/⫹299 bp promoter fragment of Flk-1 and
the 2.3-kb XhoI/BamHI fragment of the first intron as an enhancer.23
pFlkp/eGFP was generated by substitution of the LacZ sequence between
HindIII and BamHI restriction sites of pGLacZ-Flk-1 p/e with GFP
sequence (HindIII and AflII fragment) of pEGFP.N3 (Clontech, Palo Alto,
CA) (Figure 1A). The pFlkp/eGFP was linearized by PvuI digestion before
transfection into ES cells. The pPGKpuro.bpA, which carries a puromycinresistant gene, was cotransfected or, alternatively, subcloned into the SalI
site of the pFlk-1p/eGFP in a direction reverse to GFP before linearization.
For transgenic mice, the splice donor and acceptor sequences of pSV␤
(Clontech, Palo Alto, CA) were ligated to the GFP cDNA in pFlkp/eGFP.
Cell lines
EB5 (a kind gift from Dr Hitoshi Niwa, RIKEN, Kobe, Japan) is a subline
derived from E14tg2a ES cells.26 This line was generated by targeted
integration of an Oct3/4-IRES-BSD-pA vector into the Oct-3/4 allele and
carries the blasticidin S resistant selection marker gene driven by the
Oct-3/4 promoter, which is active under the undifferentiated status.27
Undifferentiated ES cells were maintained on gelatin-coated dishes in
Glasgow minimum essential medium (G-MEM; GIBCO-BRL) supplemented with 1% fetal calf serum (FCS), 10% knockout serum replacement
(KSR; GIBCO-BRL), 0.1 mM nonessential amino acids, 1 mM sodium
pyruvate (GIBCO-BRL), 0.1 mM 2-mercaptoethanol (2ME), 1000 U/mL
leukemia inhibitory factor (LIF) (GIBCO-BRL), and 20 ␮g/mL blasticidin
S to eliminate differentiated cells. OP9 stromal cell line was maintained in
alpha minimum essential medium (MEM) (GIBCO BRL) supplemented
with 20% FCS (HyClone Laboratories, Logan, UT).28
The ES cells were electroporated with linearized plasmids and then
selected for resistance to puromycin (2 ␮g/mL). In this study, 5
independent transfectants were analyzed. No substantial differences in
differentiation ability among the transfected clones and the parental cell
line were observed.
In vitro differentiation of ES cells
Induction of ES cell differentiation was carried out as described previously.19,21 Briefly, 3 ⫻ 104 undifferentiated ES cells were transferred to
each well of a type IV collagen-coated 6-well plate (BIOCOAT; Becton
Dickinson Labware, Bedford, MA) and incubated for 4 days in alpha MEM
(GIBCO BRL) supplemented with 10% FCS and 50 ␮M 2ME (induction
medium) in the absence of LIF (Figure 1B). Cultured cells were harvested
with cell dissociation buffer (GIBCO BRL) and analyzed for expression of
GFP and Flk-1 by flow cytometry. The Flk-1⫹ cells were sorted from the
harvested cells for a second round of induction. Sorted Flk-1⫹ cells
(3-10 ⫻ 104) were transferred to each well of a 6-well plate (Becton
Dickinson) that was preseeded with OP9 stromal cells. These cells were
recovered after 1 to 5 days and analyzed for expression of GFP, VEcadherin, CD45, and Ter119 by flow cytometry. The VE-cadherin⫹
CD45⫺Ter119⫺ population in the harvested cells was sorted into GFP⫹ and
GFP⫺ fractions and cultured for further induction of hematopoietic or
endothelial cells.
For the measurement of frequency of hematopoietic precursors, sorted
cells were transferred into a 6-well plate that was preseeded with OP9
stromal cells and incubated in the induction medium supplemented with a
mixture of recombinant growth factors containing 200 U/mL murine
interleukin-3 (IL-3), 2 U/mL human erythropoietin (Epo), 100 ng/mL
murine granulocyte colony-stimulating factor (G-CSF), and 100 ng/mL
murine stem cell factor (SCF). Recombinant Epo and G-CSF were
purchased from R&D Systems. Recombinant IL-3 and SCF were prepared
as previously described.29 After 24 hours, medium was replaced with a fresh
semisolid medium consisting of the induction medium, a mixture of growth
factors, and 1.2% methylcellulose (Muromachi Kagaku, Tokyo, Japan).
Cells were cultured for 6 days, and hematopoietic colonies were scored
under a microscope.
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HIRAI et al
For induction of endothelial cell growth, sorted cells were put into a
6-well plate that was preseeded with OP9 cells and incubated in the
induction medium. After 10 days, the cultures were fixed in situ with 4%
paraformaldehyde and stained with either rat anti–Flk-1 or rat anti–
PECAM-1 MoAbs. Flk-1⫹/PECAM-1⫹ endothelial cell colonies were
detected by alkaline phosphatase–conjugated antirat immunoglobulin G
(IgG) antibody (Jackson ImmunoResearch Laboratories, West Grove, PA)
and nitroblue tetrazolium (NBT)/BCIP (5-bromo-4-chloro-3-indolylphosphate) substrate solution (Boehringer Mannheim, Mannheim, Germany).
Single cell deposition assay for hemangiogenic potential
Single cell deposition of sorted cells into separate wells of 96-well plates
(Becton Dickinson) was carried out by the Clon-Cyt system of FACS
Vantage (Becton Dickinson). Sorted single cells were cocultured with OP9
stromal cells with 100 ng/mL SCF, 200 U/mL IL-3, 2 U/mL Epo, and 100
ng/mL G-CSF for 7 days. The presence of hematopoietic colonies was
judged morphologically and by May-Giemsa staining. The endothelial
colonies were immunostained with anti–PECAM-1 MoAb.
Staining of DiI-labeled acetylated low-density
lipoprotein (DiI-Ac-LDL)
Staining of DiI-Ac-LDL was performed as previously described.20 Cultured
cells were incubated in alpha MEM supplemented with 10 mg/mL
DiI-Ac-LDL (Biomedical Technologies, Stoughton, MA) in chamber slides
for 4 hours. Cells were then washed with alpha MEM and observed by
fluorescence microscopy (Axiovert 135M; Zeiss, Jena, Germany).
Transgenic mice
Linearized plasmids were purified by NACS PREPAC (GIBCO BRL),
adjusted to 5 ng/mL, and injected into mouse oocytes as described.30
Transgenic integration was confirmed by polymerase chain reaction (PCR)
of genomic DNA obtained from mouse ears. The primers used were Flk-1
p/e, 5⬘-AGTCTGTGCCTGAGAACTGG-3⬘, and GFP, 5⬘-GTAGTTGTACTCCAGCTTGTGC-3⬘. Three independent transgenic lines were established and analyzed for the expression of GFP.
Embryos were harvested at 10.5 to 12.5 days after coitus (dpc) as
described21 and subjected to immunofluorescence, FACS, or cell sorting
analyses. For immunofluorescence, embryos were fixed in 4% paraformaldehyde, embedded in optimum cutting temperature (OCT) compound, and
cryosectioned. Sections (9-12 ␮m) were stained with rabbit anti-GFP and
rat anti–PECAM-1 for primary antibodies and Alexa 488–conjugated
antirabbit IgG (H⫹L) and Alexa 564-conjugated antirat IgG (H⫹L)
(Molecular Probe) for secondary antibodies.
Reverse transcribed-polymerase chain reaction (RT-PCR)
Total RNA was prepared from sorted cells or cultured cells using ISOGEN
(Nippon Gene, Toyama, Japan). RNA was reverse-transcribed with Superscript II reverse transcriptase (GIBCO BRL) and oligo (dT)12-18 primer
(GIBCO BRL) according to the manufacturer’s instructions. PCR assays
were performed in the reaction mixture containing 1⫻ ExTaq Buffer
(Takara Shuzo, Osaka, Japan), 200 ␮M dNTPs (Pharmacia), 25 U/mL
ExTaq DNA polymerase (Takara Shuzo), several dilutions of cDNA, and 2
␮mol/L of specific primers. Sequences of primers and conditions for PCR
were described elsewhere: GATA2,31 Runx1,32 SCL,33 and c-Myb.34 PCR
products were electrophoresed through 1% agarose gel and analyzed by
staining with ethidium bromide.
sense probe. Embryos (10.5 dpc) were fixed in 4% paraformaldehyde,
embedded in OCT compound, and cryosectioned. These specimens were
fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15
minutes and then washed with PBS for 2 minutes. The sections were treated
with 7.5 ␮g/mL proteinase K in PBS at 37°C for 1 hour, washed with PBS
for 2 minutes, refixed with 4% paraformaldehyde in PBS, again washed
with PBS for 2 minutes, and placed in 0.2 M HCl for 10 minutes. After
washing with PBS for 2 minutes, the specimens were acetylated by
incubation in 0.1 M triethanolamine-HCl, pH 8.0, for 1 minute and further
in 0.1 M triethanolamine-HCl, 0.25% acetic anhydride for 10 minutes. After
washing with PBS for 2 minutes, the samples were incubated with 3%
hydrogen peroxide for 1 hour, washed in PBS for 2 minutes, and dehydrated
through a series of ethanols. Hybridization was performed with probes at
concentrations of 500 ng/mL in a hybridization solution (50% formamide,
5⫻ saline sodium citrate [SSC], 1% sodium dodecyl sulfate [SDS], 50
␮g/mL tRNA, and 50 ␮g/mL heparin) at 55°C for 16 hours. After
hybridization, the specimens were washed in 5⫻ SSC at 55°C for 15
minutes and then in 50% formamide, 2⫻ SSC at 55°C for 15 minutes,
followed by RNase treatment in 50 ␮g/mL RNase A in 10 mM Tris
(tris(hydroxymethyl)aminomethane)–HCl, pH 8.0, 1 M NaCl, and 1 mM
EDTA (ethylenediaminetetraacetic acid). Then the sections were washed
twice with 2⫻ SSC at 50°C for 15 minutes, twice with 0.2⫻ SSC at 50°C
for 15 minutes, and once with TBST (0.1% Tween 20 in Tris-buffered saline
[TBS]) for 5 minutes. After treatment with 1% blocking reagent (Roche
Diagnostics) in TBST for 1 hour, the samples were incubated with
antidigoxigenin peroxidase (POD), Fab fragment (Roche Diagnostics)
diluted 1:100 with the blocking reagent for 1 hour. The sections were
washed 3 times with 0.1% Tween 10 in PBS (PBST), incubated with
tyramide signal amplification (TSA) plus Cy3 system for coloring reaction,
and washed 3 times with PBST.
Results
Activity of Flk-1 promoter/enhancer in undifferentiated ES cells
Kappel et al23 demonstrated in a previous study that Flk-1 p/e is
active in most embryonic ECs throughout embryogenesis. Endogenous Flk-1, however, was shown to be expressed not only in ECs
but also in lateral mesoderm and some fractions of hematopoietic
cells.24,35-37 Thus, whether Flk-1 p/e accurately represents the
endogenous Flk-1 cis-regulating region or only part of its activity
remained to be investigated. To assess the activity of Flk-1 p/e in
more detail, we took advantage of the ES cell culture system that
allows detailed dissection of the differentiation process of ECs
from mesoderm (Figure 1B). For this purpose, we established ES
cell lines that were stably transduced with a GFP gene conjugated
to the Flk-1 p/e (Figure 1A).
To our surprise, all stably transformed ES cell lines expressed
GFP before induction of differentiation (Figure 2). As all independent cell lines expressed GFP, it is likely that this expression pattern
In situ hybridization
Digoxigenin-11–uridine triphosphate (UTP)–labeled single-stranded RNA
probes were prepared by using the DIG RNA labeling kit (Roche
Diagnostics, Mannheim, Germany). To generate the Runx1 probe, a
nucleotide 1-1038 fragment of Runx1 cDNA was cloned into the EcoRI and
BamHI sites of pBluescript KS⫹. This plasmid was either linearized with
XhoI and transcribed by T7 RNA polymerase to generate an antisense probe
or linearized with NotI and transcribed by T3 RNA polymerase to generate a
Figure 2. Flow cytometric analysis of the differentiating ES cells. Undifferentiated parental ES cells (top row) and ES cells transfected with GFP under the control
of Flk-1 promoter/enhancer (bottom row) were cultured on type IV collagen–coated
dishes in the absence of LIF and analyzed for the expression of Flk-1 and GFP for 4
days. The results shown are representative of 3 independent experiments.
BLOOD, 1 FEBRUARY 2003 䡠 VOLUME 101, NUMBER 3
is specific to the Flk-1 p/e rather than variations among ES cell
lines or sites to which the transgene was integrated. As Flk-1 is not
expressed in ES cells,19,24 it appears that Flk-1 p/e activity differs
from that of the endogenous regulatory regions. This ectopic
activity disappeared rapidly on induction of ES cell differentiation
by removing LIF from cultures on collagen IV-coated dishes. Of
note, however, is that GFP expression could not be detected even at
4 days of incubation under our culture conditions, although more
than 15% of cells expressed endogenous Flk-1, representing
differentiation of lateral mesodermal cells. This result indicates
again the difference between Flk-1 p/e and the endogenous
cis-regulatory region of the Flk-1 gene.
Activity of Flk-1promoter/enhancer in endothelial cells
Purified Flk-1⫹ VE-cadherin⫺ GFP⫺ mesoderm cells could rapidly
give rise to GFP⫹ cells on an OP9 stromal cell layer. To specify the
cells in which Flk-1 p/e is active, we analyzed the expression of
other surface markers in GFP⫹ cells. As shown in Figure 3A, VE
cadherin⫹ GFP⫺ cells appeared within 24 hours, followed by the
emergence of VE-cadherin⫹ GFP⫹ cells. The GFP signals were
restricted to the VE-cadherin⫹ fraction, and most GFP⫹ cells
harvested from day 3 cultures of Flk-1⫹ GFP⫺ cells coexpressed a
series of EC markers such as Flk-1, PECAM-1, and Tie-2 (Figure
3B). In addition, GFP⫹ cells formed sheetlike colonies on the OP9
feeder layer, which could be labeled by acetylated-LDL (Figure
3C). All of these observations are consistent with an idea that Flk-1
p/e is active in mature EC cells.
Figure 3. Endothelial nature of cells expressing GFP driven by the Flk-1
promoter/enhancer. (A) Sorted Flk-1⫹ cells derived from ES cells were cultured on
OP9 stromal cells and analyzed for the expression of GFP and Flk-1 on wild-type
cells (top row) or analyzed for the expressions of GFP and VE cadherin on Flk-1⫹
cells (bottom row) from day 1 to day 3. (B) After a 3-day culturing of the sorted Flk-1⫹
cells, Tie-2 and PECAM-1 expression in GFP⫹ cells were analyzed by flow cytometry.
All the GFP⫹ cells express Tie-2 and PECAM-1. (C) Incorporation of DiI-labeled
acetylated LDL (red) by GFP⫹ cells (green) was examined by fluorescent microscopy
on day 3 of Flk-1⫹ cell culture on OP9 cells. Original magnification ⫻200. The results
shown are representative of 3 independent experiments.
HEMOGENIC AND NONHEMOGENIC ENDOTHELIUM
889
Figure 4. Expression of hematopoietic markers and that of GFP were reciprocally exclusive. Sorted Flk-1⫹ cells derived from ES cells were cultured on an OP9
stromal cell layer and analyzed for the expression of hematopoietic markers (CD45
and Ter119; bottom row), an endothelial marker (VE cadherin; top row), and GFP for 5
days. The results shown are representative of 3 independent experiments.
Absence of hemogenic potential in GFPⴙ endothelial cells
Flk-1⫹ VE-cadherin⫺ GFP⫺ mesoderm cells could also give rise to
CD45⫹ or Ter119⫹ HPCs in the culture (Figure 4). Although HPCs
and GFP⫹ ECs were already present in cultures of the same stage
(from day 2 to day 5), none of the GFP⫹ cells expressed CD45 or
Ter119. During the course of this study, we noticed that sorted
GFP⫹ cells could not give rise to HPCs. It is thus likely that the
GFP⫹ population represents a subset of ECs that is excluded from
hemogenic potential. This possibility may also account for the
absence of CD45⫹ GFP⫹ cells that may represent a transitory stage
from EC to HPC, as no HPCs are generated from GFP⫹ cells. To
examine this possibility, we sorted the VE-cadherin⫹
CD45⫺Ter119⫺GFP⫺ cells (Flk-GFP⫺ ECs) and VE-cadherin⫹
CD45⫺Ter119⫺GFP⫹ cells (Flk-GFP⫹ ECs) from the culture of
Flk-1⫹ cells and assessed the frequency of precursors that give rise
to HPCs or ECs (Figure 5A-F). Except for GFP expression, the 2
populations were indistinguishable in terms of the expression of
Flk-1, VE-cadherin, PECAM, CD34, and Tie-2 (data not shown),
although it is difficult to formally rule out the possibility of
contamination of other cell lineages. Flk-GFP⫺ ECs were observed
in the culture of Flk-1⫹ VE cadherin⫺ cells from day 1 to day 4
(Figure 4) and harbor potential to give rise to definitive type HPCs
(Figure 5A,B). In contrast, neither CD45 nor Ter119 were expressed in the culture of GFP⫹ population (Figure 5A).
We also measured the frequency of clonogenic progenitors for
HPCs and ECs in each population. The frequency of cells that can
give rise to EC colonies on OP9 are nearly the same between the 2
populations (Figure 5C,D), whereas that of hematopoietic progenitors in Flk-GFP⫺ ECs is 20-fold higher than that in Flk-GFP⫹ ECs
(Figure 5E). This finding was further confirmed by single cell
deposition assay of each population into 96-well dishes that
support generation of ECs and HPCs. As shown in Figure 5F, no
hemogenic activity was observed in Flk-GFP⫹ ECs, whereas 10%
of the GFP⫺ population contained hematopoietic progenitors,
among which 3% can also give rise to ECs.
We have previously shown that ␣4-integrin is a marker for the
earliest precursor of HPC lineage derived from ECs.21 When we
analyzed the VE-cadherin⫹ cells derived from Flk-1⫹ mesodermal cells,
expression of ␣4-integrin and that of GFP were exclusively reciprocal
(data not shown). These results indicate that Flk-1 p/e activity is specific
to ECs that have already been excluded from a HPC fate.
Differential expression of transcriptional regulators
in the EC populations
All of the above-mentioned results indicate that nonhemogenic
ECs can be distinguished prospectively by the Flk-1 p/e activity.
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HIRAI et al
with the data in ES cultures, VE-cadherin⫹ cells were observed in
both GFP⫹ (16.8%) and GFP⫺ fractions (2.3%), and the number of
cells in the GFP⫺ fraction decreased in 12.5-dpc embryo (0.3%).
The expression of hematopoietic markers (CD45 or Ter119) and
GFP were exclusively reciprocal. We next sorted VE-cadherin⫹
GFP⫹ and VE-cadherin⫹ GFP⫺ populations (CD45⫺ and Ter119⫺)
from 10.5- or 11.5-dpc embryos and analyzed the ability to give
rise to HPCs or ECs (Figure 7C and Table 1). PECAM-1⫹
endothelial sheet colonies were found in wells seeded with either
GFP⫺ or GFP⫹ ECs. In complete agreement with the results
obtained from ES cell experiments, HPCs were generated only in
the cultures of GFP⫺ endothelial cells (Figure 7C).
Presence of GFPⴚ endothelial cells in the dorsal aorta
Figure 5. Hemogenic ability of GFPⴚ or GFPⴙ endothelial cells. (A) GFP⫺ or
GFP⫹ endothelial cells (VE cadherin⫹, CD45⫺, and Ter119⫺) were harvested from
2.5-day culture of sorted Flk-1⫹ cells and cultured on OP9 cells. Then the expression
of hematopoietic markers and GFP were analyzed by flow cytometry 24 hours later.
The result shown is a representative of 3 independent experiments. (B) May-GrünwaldGiemsa staining of hematopoietic cells formed in the culture of GFP⫺ endothelial
cells. Erythroblasts, monocytes, macrophages, and polymorphonuclear cells were
observed. The scale bar represents 10 ␮m. (C) Morphology of a PECAM-1⫹ colony
generated from either GFP⫺ or GFP⫹ endothelial cells. The scale bar represents 200
␮m. (D) Frequency of cells capable of formation of endothelial colony in the indicated
fractions. Error bars indicate standard deviations for 3 independent determinations.
(E) Frequency of hematopoietic colony-forming cells in the indicated fractions. Error
bars indicate standard deviations for 3 independent determinations. (F) Incidence of
hematopoietic and endothelial cell differentiation from single GFP⫺ or GFP⫹
endothelial cells (VE cadherin⫹, CD45⫺, and Ter119⫺) on OP9 stromal cell layer in
the presence of SCF, IL-3, Epo, and G-CSF. Arrows indicate that no colony-forming
cell was detected. Error bars indicate standard deviations for 3 independent
determinations.
Thus, it is interesting to assess the expressions of molecules such as
Runx1, c-Myb, SCL, and GATA2 that have been shown to play a
requisite role in HPC differentiation.32,38-40
VE-cadherin⫹ cells were divided into GFP⫹ and GFP⫺ populations, and the expression of various genes was assessed by RT-PCR
(Figure 6). Expressions of Runx1 and c-Myb that are essential for
the differentiation of the definitive HPCs were detected preferentially in GFP⫺ population, whereas only low-level expression was
observed in GFP⫹ population. In contrast, both populations expressed GATA2 and SCL at equal levels.
To determine the regions in the embryos where GFP⫺ ECs exist, we
sectioned 10.5-dpc transgenic embryos and stained them with anti-GFP
(green) and anti–PECAM-1 antibodies (red). As previously reported,23
GFP was expressed by nearly all the endothelial cells (Figure 8A-C).
PECAM-1⫹ endothelial cells expressed GFP, and, in addition, there
were PECAM-1⫹ round cells circulating within the vessels but were
negative for GFP. These findings corroborate with the observation from
ES cell experiments. Outside the endothelium, GFP was expressed only
in a limited portion of the neural tube (data not shown). Taking into
consideration the fact that GFP⫺ ECs contain hemogenic ECs, we
analyzed GFP expression in dorsal aorta that has been implicated as the
site of HPC generation from ECs. As shown in Figure 8D-L, integration
of GFP⫺ endothelial cells was observed in the ventral wall of the dorsal
aorta, where hematopoietic clusters were formed, and the rest of the ECs
were GFP⫹ (Figure 8D-L). It was recently shown that a subset of
endothelium that expresses Runx1 includes a potent progenitor for
definitive hematopoiesis.41 By in situ hybridization, we also confirmed
that expression of Runx1 was observed at the cell clusters on the ventral
wall of the dorsal aorta in 10.5-dpc embryo, whereas other cells of inner
linings of blood vessels were negative for the transcripts (Figure 8M). To
show that Runx1 expression is exclusive to Flk-GFP, we sorted GFP⫹
and GFP⫺ endothelial cells from 10.5-dpc transgenic mouse and
analyzed the expression of Runx1 (Figure 8N). Consistent with the
results in ES cell experiments, Runx1 was preferentially expressed by
Flk-GFP–negative endothelial cells. These data show that Runx1⫹
endothelial cells are integrated within the ventral wall of dorsal aorta and
they are negative in GFP.
Discussion
Expression of Flk-1 has been evaluated by using MoAb and mice in
which marker genes such as LacZ were knocked into the Flk-1
Flk-1 promoter/enhancer activity in the embryo
Given that GFP expression can specify the ECs that are excluded
from HPC fate, it is of great interest to determine the regions in the
embryo where GFP⫹ and GFP⫺ ECs are present. For this purpose,
we generated transgenic mouse strains harboring the same construct as used in the ES experiment. As previously reported, nearly
all the vessels expressed GFP in developing embryos (Figure 7A).
To correlate the data of the transgenic mice with that of ES
cell-derived cells, we dissociated 10.5-dpc embryos and analyzed
the expression of GFP by flow cytometry (Figure 7B). Consistent
Figure 6. mRNA expression of transcription factors. Flk-1⫹ VE cadherin⫺ cells,
GFP⫺ VE cadherin⫹ CD45⫺Ter119⫺ cells, and GFP⫹ VE cadherin⫹ CD45⫺Ter119⫺
cells were induced in vitro from ES cell-derived Flk-1⫹ cells for 2.5 days, and different
dilutions of cDNA prepared from sorted cells were subjected to PCR amplification
using primers specific for Runx1, SCL, c-Myb, GATA-2, and ␤-actin. PCR products
were separated on 1% agarose gel and stained with ethidium bromide. The results
shown are representative of 3 independent experiments.
BLOOD, 1 FEBRUARY 2003 䡠 VOLUME 101, NUMBER 3
HEMOGENIC AND NONHEMOGENIC ENDOTHELIUM
891
Figure 7. Activity of the Flk-1 promoter/enhancer in vivo. (A) Direct fluorescent image of a 10-dpc mouse embryo transgenic for a reporter gene construct in which the GFP
gene is under the control of the Flk-1 promoter/enhancer. (B) Expression of VE cadherin (top row) and GFP in CD45⫺, Ter119⫺ (bottom row), and GFP and hematopoietic
markers in cells dissociated from 10.5- and 12.5-dpc transgenic mouse embryos. The result shown is a representative of 3 independent experiments for 3 independent clones.
(C) Morphology of hematopoietic and PECAM⫹ endothelial colonies generated from either GFP⫺ endothelial cells obtained from 10.5-dpc transgenic embryos. Original
magnification ⫻100.
locus.24,42,43 Previous results indicated that Flk-1 expression is
detected during successive stages from early lateral mesoderm to
endothelial stages.19 Moreover, it was also reported that Flk-1
expression is maintained in nascent HPCs for a short interval after
differentiation from Flk-1⫹ progenitors.37 In contrast to commonly
used endothelial markers such as Tie-2 or CD34 that are expressed
also in adult hematopoietic stem cells,44,45 Flk-1 expression is
unique in that it is restricted to mesodermal cells and ECs and is
absent in adult HPCs. Kappel et al23 investigated the cis-regulatory
region of the Flk-1 gene and showed that a combination of a
5⬘-flanking promoter region together with the 3⬘ portion of the first
intron, Flk-1 p/e, can direct gene expression specifically in the EC
population in the embryo after 7.8-dpc. Although the activity of
Flk-1 p/e decreases in the ECs of adult mice, it appears again in the
ECs during neoangiogenesis.46 Although these studies suggested
that Flk-1 p/e can largely dictate the expression pattern of the
endogenous Flk-1 gene, it remained unclear whether it is also
active in the Flk-1⫹ nonendothelial populations such as lateral
mesoderm and also nascent HPCs.
In this study, using both the ES cell differentiation system and
transgenic mice, we showed that the Flk-1 p/e is not identical to the
endogenous regulatory unit of the Flk-1 gene. First, it is active in
undifferentiated ES cells, although endogenous Flk-1 expression is
not detectable in ES cells either by flow cytometry with anti–Flk-1
MoAb or RT-PCR analyses.19,37 The expression level of GFP driven
by this promoter is fairly strong and found in all 5 independent ES
cell lines, indicating that the observed activity represents an
autonomous activity of Flk-1 p/e rather than a reflection of
integrated sites. Of note is that this activity is specific to LIF-
maintained ES cells, as its activity disappears rapidly on removal of
LIF. This ectopic activity of Flk-1 p/e indicates the presence of
additional cis-regulatory elements outside this region, which
repress Flk-1 gene activation in ES cells. The second difference
between Flk-1 p/e and the endogenous cis-regulatory region is that
the former is not active in Flk-1⫹ lateral mesoderm cells and Flk-1⫹
hematopoietic cells, although the endogenous Flk-1 gene is continuously expressed throughout these successive stages. Activation of
Flk-1 p/e occurs only after the differentiation of Flk-1⫹ VEcadherin⫺ lateral mesoderm to Flk-1⫹ VE-cadherin⫹ cells. This
result is interesting in that it demonstrates that Flk-1 gene
expression is regulated differently even in the successive stages
during which Flk-1 expression is maintained at the same level. The
shift of gene regulatory units from mesodermal type to EC type
appears to be associated with the process of EC commitment from
lateral mesoderm. However, it should be noted that GFP⫺ ECs exist
and are maintained for several days after formation of the vascular
network in the embryos, although their proportion decreases as
embryogenesis progresses. Thus, we want to view this process as
generating a new EC population in which Flk-1 p/e is active during
the process of EC maturation.
Accumulating data indicated that ECs are a highly diverse
population, which is defined by morphology, anatomical location,
differential expression of molecules, and also functional properties.
We and others have proposed that the ability to give rise to HPCs is
a way of defining EC diversity.17,18 This study demonstrated that
HPCs are generated only from a Flk-1 p/e inactive population.
Interestingly, Flk-1⫹ mesoderm contains a direct precursor of
HPCs, although its differentiation is restricted to the primitive
Table 1. Hemogenic ability of VE cadherinⴙ (CD45ⴚ Ter119ⴚ) cells obtained from transgenic embryos
No. of positive wells/no. of wells analyzed
VE cadherin⫹ GFP⫺
Embryo
VE cadherin⫹ GFP⫹
No. of cells/well
HPC colonies
EC colonies
Both
HPC colonies
EC colonies
Both
Exp 1
30
2/6
4/6
1/6
0/6
4/6
0/6
Exp 2
50
4/6
4/6
3/6
0/6
6/6
0/6
Exp 1
15
2/4
2/4
1/4
0/4
3/4
0/4
Exp 2
30
9/12
10/12
8/12
0/9
8/9
0/9
10.5-dpc
11.5-dpc
Indicated numbers of cells were sorted from embryos transgenic for GFP gene under the control of Flk-1 promoter/ enhancer and cocultured with OP9 stromal cells with
SCF, IL-3, Epo, and G-CSF for 10 days. Existences of hematopoietic colonies were judged by their morphology, and those of endothelial colonies were by immunostaining with
anti-PECAM-I MoAb. Exp, experiment.
892
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HIRAI et al
Figure 8. Localization of GFPⴚ and GFPⴙ endothelial cells in mouse embryos.
E10.5 transgenic embryos were stained with anti-GFP (green) and anti–PECAM-1
(red) antibodies. Nearly all the PECAM-1⫹ endothelial cells express GFP, and, in
addition, there were PECAM-1⫹ round cells circulating within the vessels, but they
were negative for GFP (A-C). (D-L) GFP⫺ endothelial cells (arrowheads) were found
integrated in the endothelium at the ventral wall of the dorsal aorta, where
hematopoietic clusters were formed, whereas the rest of the ECs were GFP⫹. (M-N)
expressions of the transcripts for Runx1 were shown by using in situ hybridization (M)
or RT-PCR (N). In situ hybridization was performed as described in “Materials and
methods,” and red dots indicate the expression of Runx1. The sense probe did not
give any signal (data not shown). White-dotted line indicates the outlines of dorsal
aorta (M). (N) GFP⫺ VE cadherin⫹ CD45⫺Ter119⫺ cells and GFP⫹ VE cadherin⫹
CD45⫺Ter119⫺ cells were sorted from 10.5-dpc transgenic embryos and subjected to
RT-PCR using primers specific for Runx1. PCR products were separated on 1%
agarose gel and stained with ethidium bromide. The result shown is representative of
3 independent experiments. DA, dorsal aorta.
HPCs.22 Thus, both before and after expression of VE-cadherin,
Flk-1 p/e activity is correlated inversely with the ability to generate
HPCs. The same results were obtained by sorting GFP⫹ and GFP⫺
ECs from transgenic mice harboring Flk-1 p/e–GFP gene, indicating that this is unlikely because of an in vitro artifact.
The inverse relationship between hemogenic potential and
Flk-1 p/e activity was also suggested by the differential expression
of transcriptional regulators that are expressed in both HPCs and
ECs and are essential for HPC differentiation. Our present study
showed that Runx1 and c-Myb are expressed preferentially in the
GFP⫺ population, whereas SCL and GATA2 are expressed in both
populations. Previous gene targeting studies demonstrated that the
former 2 are involved in the process of generating the definitive
HPCs. Moreover, we recently demonstrated that Runx1 is required
for the differentiation process of VE-cadherin⫹ cells to HPCs.47
Accepting the hypothesis that ECs are the precursors of the
definitive HPCs, the restriction of Runx1 and c-Myb to GFP⫺ ECs
suggests that Flk-1 p/e activation may reflect the process to
generate fully committed ECs through exclusion of hemogenic
potential. On the other hand, SCL and GATA2 were shown to have
roles in vascular development. By mutational analysis, binding
sites for SCL, GATAs, and Ets transcription factors were identified
as critical elements for the EC specificity of the Flk-1 p/e.48
However, as GATA2 and SCL are expressed equally in both GFP⫹
and GFP⫺ populations, there should be involvement of additional
molecules and sequences for regulation of the restricted expression
of Flk-1p/e in nonhemogenic ECs.
Nonetheless, Flk-1 p/e allows us to distinguish prospectively
the nonhemogenic ECs in the embryonic tissues. Previous histologic studies demonstrated that transition of ECs to HPCs is
observed most consistently in the dorsal aorta. Moreover, expression of Runx1 that is essential for this process is observed in this
region (Figure 8M and North et al41,49). Our study showed that
GFP⫺ ECs are integrated into the EC sheet that lines the luminal
wall of the dorsal aorta, indicating that both hemogenic and
nonhemogenic VE-cadherin⫹ cells are indeed an integral part of the
EC lining rather than existing separately in different tissues.
Consistent with our expectation, many HPC clusters were found
associated with ECs that were GFP⫺ but rarely GFP⫹ ECs. As only
GFP⫺ ECs had potential to give rise to HPCs, it is likely that the
HPC clusters attaching to the luminal wall of dorsal aorta are
indeed the cells generated de novo from the GFP⫺ fraction of ECs.
In conclusion, Flk-1 gene regulation appears to be under a
complex control mechanism and different sets of molecules.
Additional cis-regulatory regions that dictate the endogenous Flk-1
expression remain to be identified. As revealed in this study,
however, the Flk-1 p/e identified by Kappel et al,23 although
representing only a part of the Flk-1 regulatory region, is useful
both for dissecting the process of EC commitment and as a tool for
gene transduction to fully committed ECs.
Acknowledgments
We thank Mariko Moriyama (Riken, Center for Developmental
Biology, Kobe, Japan) for her critical help in the in situ hybridization procedure and Dr Ruth Yu for a critical reading of the
manuscript.
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