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Prepublished online as a Blood First Edition Paper on September 19, 2002; DOI 10.1182/blood-2002-02-0655.
HEMATOPOIESIS
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.
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 5'-flanking region and 3' portion of the first intron of
the Flk-1 gene (Flk-1 p/e) that has been implicated in endothelium-specific 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-1+ lateral mesoderm. Flk-1 p/e becomes
active after generation of vascular endothelial
(VE)-cadherin+ ECs. Emergence of GFP 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 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 EC-specific 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.
Monoclonal antibodies (MoAbs), cell staining, and sorting
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 µg/mL propidium iodide (PI;
Sigma) to exclude dead cells. Cells were analyzed and sorted by
fluorescence-activated cell sorter (FACS) Vantage (Becton
Dickinson Immunocytometry Systems, San Jose, CA). Data were analyzed
using the software CellQuest (Becton Dickinson Immunocytometry Systems).
Plasmids
Cell lines
The ES cells were electroporated with linearized plasmids and then selected for resistance to puromycin (2 µg/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 µM 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, VE-cadherin, 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. 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 µm) 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 µM dNTPs (Pharmacia), 25 U/mL ExTaq DNA polymerase (Takara Shuzo), several dilutions of cDNA, and 2 µmol/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.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 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 µg/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 µg/mL tRNA, and 50 µg/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 µg/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.
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 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.
Absence of hemogenic potential in GFP+ 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+
CD45Ter119GFP cells
(Flk-GFP ECs) and VE-cadherin+
CD45Ter119GFP+ 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 We have previously shown that 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. 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.32,38-40VE-cadherin+ cells were divided into GFP+ and
GFP
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 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 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.
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 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+ VE-cadherin 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 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 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 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 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.
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.
Submitted February 28, 2002; accepted September 9, 2002.
Prepublished online as Blood First Edition Paper, September 19, 2002; DOI 10.1182/blood-2002-02-0655.
Supported by grants from the Ministry of Education, Culture, Sports, Science 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.
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.
Reprints: Hideyo Hirai, Department of Microbiology, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan; e-mail: hhirai{at}basic.kpu-m.ac.jp.
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G. Dai, H. Sakamoto, Y. Shimoda, T. Fujimoto, S.-I. Nishikawa, and M. Ogawa Over-expression of c-Myb increases the frequency of hemogenic precursors in the endothelial cell population. Genes Cells, August 1, 2006; 11(8): 859 - 870. [Abstract] [Full Text] [PDF]
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A. H. Licht, F. Muller-Holtkamp, I. Flamme, and G. Breier Inhibition of hypoxia-inducible factor activity in endothelial cells disrupts embryonic cardiovascular development Blood, January 15, 2006; 107(2): 584 - 590. [Abstract] [Full Text] [PDF]
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S. Tada, T. Era, C. Furusawa, H. Sakurai, S. Nishikawa, M. Kinoshita, K. Nakao, T. Chiba, and S.-I. Nishikawa Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture Development, October 1, 2005; 132(19): 4363 - 4374. [Abstract] [Full Text] [PDF]
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H. Hirai, I. M Samokhvalov, T. Fujimoto, S. Nishikawa, J. Imanishi, and S.-I. Nishikawa Involvement of Runx1 in the down-regulation of fetal liver kinase-1 expression during transition of endothelial cells to hematopoietic cells Blood, September 15, 2005; 106(6): 1948 - 1955. [Abstract] [Full Text] [PDF]
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H. Hisatsune, K. Matsumura, M. Ogawa, A. Uemura, N. Kondo, J. K. Yamashita, H. Katsuta, S. Nishikawa, T. Chiba, and S.-I. Nishikawa High level of endothelial cell-specific gene expression by a combination of the 5' flanking region and the 5' half of the first intron of the VE-cadherin gene Blood, June 15, 2005; 105(12): 4657 - 4663. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-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.
4-integrin is a marker for the
earliest precursor of HPC lineage derived from ECs.
