© 2001 American Heart Association, Inc.
Molecular Medicine |
ELF-1 Is a Transcriptional Regulator of the Tie2 Gene During Vascular Development
From the Cardiology Division (A.D., J.G., S.R., P.O.), Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass; New England Baptist Bone and Joint Institute (A.D., J.G., S.R., T.A.L., P.O.), Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass; and Department of Molecular Cell and Developmental Biology (S.T., L.I.-A.), University of California at Los Angeles, Los Angeles, Calif.
Correspondence to Peter Oettgen, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115. E-mail joettgen{at}caregroup.harvard.edu
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Abstract—Vascular development requires the tightly coordinated expression of several growth factors and their receptors. Among these are the Tie1 and Tie2 receptors, which are almost exclusively endothelial cell–specific. The critical transcriptional regulators of vascular-specific gene expression remain largely unknown. The Ets factors are a family of evolutionarily conserved transcription factors that regulate genes involved in cellular growth and differentiation. We have recently shown that the Ets factor NERF is a strong transactivator of the Tie1 and Tie2 genes. To extend these studies, we have begun to identify the Ets factors that are expressed in developing blood vessels of the chicken chorioallantoic membrane (CAM), a highly vascular embryonic network. RNA was extracted from microdissected CAM blood vessels, and reverse transcriptase–polymerase chain reaction was performed using oligonucleotides encoding conserved amino acids within the Ets domain. One of the polymerase chain reaction fragments was subcloned and identified as the chicken homologue of the Ets factor ELF-1, cELF-1. ELF-1 is most closely related to the Ets factor NERF. In situ hybridization and immunohistochemistry demonstrate that cELF-1 is enriched in developing chicken blood vessels. cELF-1 is also a strong transactivator of the Tie1 and Tie2 genes and can bind to conserved Ets sites within the promoters of these genes. A complex of similar size forms when gel shifts are performed with cellular extracts derived from the CAM blood vessels, which is recognized by an antibody against cELF-1. In summary, ELF-1 belongs to a subset of Ets factors that regulate vascular-specific gene expression during blood vessel development.
Key Words: vascular development • angiogenesis • Tie2 gene • gene expression
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Introduction |
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Vasculogenesis, the development of new blood vessels, begins during the second week of normal human embryogenesis. The Tie1 and Tie2 genes are one family of endothelial-specific receptor tyrosine kinases that have been determined to be critical for vascular development.1 They are expressed predominantly on endothelial cells of the developing vasculature. Targeted disruption of Tie1 leads to the development of leaky blood vessels, resulting in edema and hemorrhage, whereas disruption of Tie2 leads to the formation of dilated blood vessels with abnormalities in sprouting and branching of the developing blood vessels, resulting in defects in capillary network formation and early embryonic death.2 Mutations in the Tie2 gene have been identified in humans, resulting in venous malformations.3 Although these receptors were described as being completely endothelial cell–specific, it has recently been shown that both receptors are expressed in up to 30% of undifferentiated hematopoietic stem cells and 10% of B cells, suggesting a possible role in hematopoiesis in addition to vasculogenesis.4 5
The Ets genes are a family of at least 30 members that function as transcription factors.6 All Ets factors share a highly conserved 80- to 90-amino acid–long DNA-binding domain, the Ets domain. Ets factors play a central role in regulating genes involved in development, cellular differentiation, and proliferation. Many genes specific to macrophages, B cells, and T cells are regulated by Ets factors. The role of Ets factors in the immune system has been substantiated by experiments in mice, where the genes encoding several Ets factors have been disrupted by homologous recombination. The PU.1 knockout is characterized by a lack of immune-system development.7 The Ets-1 knockout mice are characterized by T-cell apoptosis and increased terminal B-cell differentiation.8
Interestingly, the main regulatory elements found thus far in the Flt-1, Tie1, and Tie2 genes contain several conserved putative Ets-binding sites that are critical for the transcriptional activity of the promoters and enhancers of these genes.9 10 11 For example, a mutation of one Ets-binding site in the promoter of the Flt-1 gene leads to a 90% reduction in the basal activity of the promoter. Likewise, in transgenic animals in which LacZ expression is directed throughout the vasculature by the Tie2 promoter and enhancer, a mutation in an Ets-binding site in the core enhancer leads to a marked reduction in vascular-directed LacZ gene expression. We have recently identified conserved Ets-binding sites in the Tie1 and Tie2 promoters, which are necessary for vascular-specific gene expression.12 13 It is presently not known which of the Ets factors are critical for the transcriptional activity of these genes.
To determine which of the Ets transcription factors are expressed during blood vessel formation and may regulate vascular-specific genes during this process, we examined developing blood vessels in the chorioallantoic membrane (CAM) of the chicken and identified the chicken homologue of ELF-1 (cELF-1). Our results demonstrate that cELF-1 is a strong transactivator of the Tie1 and Tie2 genes, can bind to specific Ets sites within the Tie1 and Tie2 promoters, and is enriched in developing blood vessels, suggesting that it contributes to the transcriptional regulation of vascular development.
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Materials and Methods |
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Cell Culture
Human umbilical vein endothelial cells (HUVECs), human dermal microvascular endothelial cells, and human aortic endothelial cells (HAECs) were obtained from Clonetics. PY41 endothelial cells were a generous gift from Vicki Bautch (University of North Carolina).14 The murine yolk-sac endothelial cell line (C166) and the EOMA cells were a generous gift from Robert Auerbach (University of Wisconsin) and were grown as previously described.15 16
RNA Extraction and Northern Blot
Analysis
Total RNA was extracted from CAM blood vessels,
cultured CAM endothelial cells, and blood derived from the CAM at
different developmental stages, as previously
described.17 Total RNA was
electrophoresed and transferred onto a Nytran membrane. The filters
were blocked in prehybridization solution and then hybridized with a
cELF-1–specific probe. The cELF-1 cDNA fragment used to generate the
probe is 700 bp in length and encodes the first 100 amino acids of the
cELF-1 protein and 100 bp of the 5' untranslated region. The size of
the band detected by Northern blot analysis is 3.4 kb. To normalize for
loading and transfer efficiency, the membranes were rehybridized with a
probe for the 36B4 chicken housekeeping gene. The size of the band
detected by Northern blot analysis is 1 kb.
Reverse Transcriptase–Polymerase Chain
Reaction and Chicken 
-Phage Library Screen
To identify Ets factors that are expressed in the
developing blood vessels of the chicken CAM, reverse
transcriptase–polymerase chain reaction (RT-PCR) was performed using
RNA extracted from the CAM blood vessels. cDNA was generated from 2
µg of total RNA by using random hexamer priming. Degenerate
oligonucleotides corresponding to conserved regions within the
Ets-DNA–binding domain were use as previously
described.18 PCR fragments
were subcloned, and fragments of the expected sized were sequenced. A
5-day chicken yolk-sac library (Stratagene) was plated and screened
with a partial cDNA fragment for cELF-1. Two full-length cDNA clones
were isolated. For RT-PCR of the human and mouse endothelial cells, the
ELF-1–specific primers used, which recognize both mouse and
human ELF-1, were 5'-ATGGCTGCTGTTGTCCAAC-3' and
5'-CCTGAGTGCTCT(C/T)CCCAT-3', with an expected amplification product of
700 bp. The GAPDH primers used were 5'-CAAAGTTGTCATGGATGACC-3' and
5'-CCATGGAGAAGGCT- GGGG-3', with an expected amplification product
of 200 bp. PCRs were performed as previously
described.19
In Situ Hybridization
Whole-mount in situ hybridization on embryonic day 3
(E3) chick embryos and E10 CAMs was carried out as described by
Wilkinson et al.20 The sense
and antisense probes were derived from the same 700-bp fragment that
was used for Northern blot analysis, which was subcloned into the PCRII
vector (Invitrogen) containing both a T7 (sense) and an Sp6 (antisense)
promoter. Briefly, embryos were fixed, dehydrated, and rehydrated
through a methanol series and washed in 1x PBT (PBS plus 0.1%
Tween-20). Embryos were then permeabilized at room temperature. After
color developed to the appropriate intensity, specimens were washed
several times and then rehydrated through the graded methanol baths.
Images of the embryos suspended in 80% glycerol were obtained using a
3CCD Toshiba camera on a Nikon SMZ-U dissecting microscope.
Digoxigenin-labeled RNA probes were prepared per the manufacturer’s
recommendations (Roche). The level of digoxigenin incorporation was
assessed by using a dot-blot comparison to a standard
(Roche).
Immunohistochemistry
Paraffin-embedded E4 and E5.5 chicken embryos were
stained with a rabbit polyclonal anti–cELF-1 antibody. Sections were
clarified with xylene and rehydrated through a decreasing gradient of
ethanol. After several washes with ddH2O
and 1x PBS, sections were treated with 0.1 mg/mL proteinase K in 1x
PBS. Hybridization with an anti–cELF-1 probe was performed overnight
after blocking for 1 hour with 2% goat serum in 1x PBT (PBS
containing 0.05% Tween-20). Sections were then incubated with
biotinylated anti-rabbit (Vector Laboratory) and fluorescein avidin DN
(Vector Laboratory), consecutively. Immunostained sections were then
analyzed by confocal microscopy. Nuclear staining was made possible
with the addition of 0.1 mg/mL propidium iodide dissolved in 1:1
PBS/glycerol mounting medium.
DNA Transfection Assays
Cotransfections of 1.5 to
2x105 endothelial cells or 293 HEK cells
were performed using 1.75 µg of the reporter-gene construct DNA and
0.75 µg of the expression-vector DNA with Lipofectamine (Gibco BRL).
The cells were harvested 16 hours after transfection and assayed for
luciferase. Individual transfections were performed in duplicate and
repeated independently in triplicate with similar results.
Cotransfection of a second plasmid for determination of transfection
efficiency was omitted, because potential artifacts with this technique
have been reported21 and
because many commonly used viral promoters contain potential binding
sites for Ets factors.
In Vitro Transcription-Translation
Full-length chicken and human ELF-1 cDNA encoding the
entire open-reading frames were inserted downstream of the T7 promoter
into the Bluescript vector. Coupled in vitro transcription–in vitro
translation reactions were performed with 1 µg of plasmid DNA using
the TNT reticulocyte lysate kit (Promega) and T7 RNA polymerase as
recommended by the manufacturer. The plasmid vector without an insert
was used as a control.
Electrophoretic Mobility Shift Assay
DNA-binding reactions were performed as previously
described.18 22
Briefly, 20-µL samples containing 2 µL of in
vitro–translated products or cell extracts were incubated with a
solution containing 32P-labeled
double-stranded probes (30 000 cpm). Samples were incubated in the
presence or absence of increasing amounts of cold competitor (5 or 50
ng) for 15 to 20 minutes at room temperature and run on a 4%
polyacrylamide gel (acrylamide-bisacrylamide, 29:1) containing a buffer
of 0.25x TBE (22.5 mmol/L Tris borate and 0.5 mmol/L EDTA).
Oligonucleotides used as probes and for competition studies were as
follows:
Tie2 promoter oligonucleotide
5'-TGCAAAGGAAACAGGAAAAAGGAACTTAAC-3'
3'-ACGTTTCCTTTGTCCTTTTTCCTTGAATTG-5'
Tie1 P1
5'-ACTGGCTTCCTCCCTTTCCTGTCTC-3'
3'-TGACCGAAGGAGGGAAAGGACAGAG-5'
Tie1 P2
5'-CCATCATTTCCTCTTCCTCCCCAG-3'
3'-GGTAGTAAAGGAGAAGGAGGGGTC-5'
Tie1 P2 Mut1
5'-CCATCATTTAATCTTCCTCCCCAG-3'
3'-GGTAGTAAATTAGAAGGAGGGGTC-5'
Tie1 P2 Mut2
5'-CCATCATTTCCTCTTAATCCCCAG-3'
3'-GGTAGTAAAGGAGAATTAGGGGTC-5'
Tie1 P2 Mut1,2
5'-CCATCATTTAATCTTAATCCCCAG-3'
3'-GGTAGTAAATTAGAATTAGGGGTC-5'
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Isolation of the cELF-1
In an effort to identify transcription factors belonging to the Ets family that are expressed during blood vessel development, we chose to examine the highly vascular chicken CAM. Vessels within the CAM undergo an exponential growth followed by a quiescent phase. Total RNA was extracted from blood vessels that were microdissected from 10-day-old CAMs. RT-PCR was performed with degenerate PCR primers corresponding to conserved regions of the Ets domain, allowing the identification of a partial DNA sequence for a member of the Ets-factor family. This fragment was used to screen a chicken yolk-sac cDNA library and isolate a cDNA clone encoding the full-length cELF-1. As shown in Figure 1
, cELF-1 encodes a 617-amino acid–long protein with
an expected molecular mass of 71.0 kDa. The highest degree of homology
to ELF-1 exists in the DNA-binding domain (100%), with overall protein
sequence homology of 77%. We have previously identified additional
regions of homology between ELF-1 and a closely related Ets factor,
NERF, in the transactivation domain. These 4 domains (A through D) are
also highly conserved between human, murine, and chicken
ELF-1.23 To demonstrate that
translation of the cELF-1 generates a protein of the expected size, we
performed in vitro transcription translation with
35S-methionine. As shown in
Figure 2, translation of the cELF-1 and human ELF-1 cDNA
fragments generated fragments of the expected
size.
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Expression Pattern of cELF-1 in the Chicken
CAM
To determine the expression pattern of cELF-1 in the
CAM, Northern blot analysis was performed using RNA derived from CAM
blood vessels at different developmental stages. As shown in
Figure 3A, cELF-1 is highly expressed in the CAM blood
vessels. Because it has previously been shown that ELF-1 is expressed
in T and B cells, we examined the expression of cELF-1 in fetal chicken
blood at different stages of development. As expected, cELF-1 is also
highly expressed in chicken blood
(Figure 3B). To ascertain whether cELF-1 is expressed in the
CAM blood vessels devoid of blood, RNA was extracted from CAM blood
vessels flushed free of blood and from unflushed CAMs. Although
flushing the blood vessels diminishes the expression of cELF-1, there
is still significant expression of cELF-1 in the flushed vessels
(Figure 3C). Furthermore, cELF-1 expression is demonstrated
in fetal chicken endothelial cells, compared with chicken fibroblasts,
which demonstrate minimal expression of cELF-1.
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ELF-1 Is Expressed in a Subset of Human and
Murine Endothelial Cells
We were somewhat surprised to find cELF-1 highly
expressed in the CAM blood vessels, because we had previously examined
ELF-1 expression in human endothelial cells and not detected it in
either HUVECs or HAECs.13 To
extend these studies, we examined additional murine and human
endothelial cells for the expression of ELF-1. As shown in
Figure 3D
, ELF-1 is also expressed in the murine yolk-sac
endothelial line C166, EOMA and PY41 endothelioma lines, and human
dermal microvascular endothelial cells. This suggests that ELF-1 is
only expressed in a subset of endothelial cells. As a positive control,
we used the B-cell lines A20 and HAFTL, which we have previously shown
express
ELF-1.23
Expression Pattern of cELF-1 in the
Embryo
We have previously demonstrated that human ELF-1 is
highly expressed in several fetal tissues, including the heart and
liver, and, weakly, in the
brain.23 To ascertain cELF-1
expression at different developmental stages, Northern blot analysis
was performed with chicken fetal organs at different developmental
stages. As shown in
Figure 4, cELF-1 is strongly expressed in the fetal liver,
in several later developmental stages in the heart, and in a temporal
window in embryonic brain and limb development.
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In Situ Hybridization of cELF-1 in the
Developing Blood Vessels of the Chicken CAM
Having demonstrated strong expression of cELF-1 in the
CAM blood vessels at different stages by Northern blot analysis, the
expression of cELF-1 was examined by in situ hybridization to
additionally define the expression pattern of cELF-1 during blood
vessel development. As shown in
Figure 5A (top), cELF-1 is expressed along the lining of the
larger caliber blood vessels, with a punctate expression pattern in the
smaller branching vessels. At higher magnification, strong expression
of cELF-1 is demonstrated in these smaller-caliber branching vessels
(Figure 6A, HP). Whole-mount in situ hybridization
also confirmed strong expression in the developing heart at day 10
(Figure 5B).
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Immunohistochemistry of cELF-1 in the
Developing Chicken Embryo
To examine cELF-1 protein expression during chicken
embryogenesis, with a particular focus on blood vessel development,
immunohistochemistry was performed using an ELF-1 polyclonal antibody.
Paraffin-embedded sections of E4 and E5.5 chicken embryos were
incubated with anti–cELF-1, and immunocomplexes were detected with
biotinylated anti-rabbit antibody followed by fluorescein avidin DN.
Visualization of nuclei was performed with propidium iodide. As shown
in
Figure 6A, cELF-1 is highly expressed in the inner lining of
the developing dorsal aorta (DA) of E4 chicken embryos. At higher
magnification
(Figure 6B), cELF-1 expression is also appreciated in a
subset of blood cells within the lumen of the aorta, as would be
expected, because ELF-1 has previously been shown to be expressed in a
subset of hematopoietic cells. No expression could be detected with
preimmune serum
(Figures 6C and 6D). cELF-1 was also detected in smaller
developing chicken blood vessels
(Figure 6E) and in intersomitic vessels of the 4-day chicken
embryo
(Figure 6F). Because we detected high levels of cELF-1
expression in the developing chicken heart by whole-mount in situ
hybridization, we also examined cELF-1 protein expression in the
developing heart. At lower magnification
(Figure 6G), expression is appreciated in the heart but not
the surrounding lung. At higher magnification, cELF-1 expression is
detected in the endocardium of the heart
(Figure 6H). Interestingly, expression was also detected on
the pericardial surface.
cELF-1 Can Transactivate the Tie1 and Tie2
Promoters
We have previously shown that one of the NERF
gene isoforms, NERF2, is a strong transactivator of the Tie2
gene.13 Because ELF-1 is
highly homologous to NERF2, we tested the ability of the chicken and
human forms of ELF-1 to transactivate the Tie2 gene promoter. As shown
in
Figure 7A, cELF-1 was similar to human ELF-1 in its ability
to transactivate the Tie2 promoter, in contrast to Ets-1 and Ets-2,
which only weakly transactivate the Tie2 promoter. Because we have
previously demonstrated that the Ets factor NERF is similarly able to
transactivate the Tie1 gene, we were interested in testing whether the
human or chicken ELF-1 could similarly transactivate the Tie1
promoter.12 As shown in
Figure 7B, both chicken and human ELF-1 are strong
transactivators of the Tie1 promoter. This suggests that both the Tie1
and Tie2 genes may be gene targets for ELF-1.
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cELF-1 Can Bind to Ets Sites in the Tie1
and Tie2 Promoter
We have previously identified the Ets-binding
sites that are necessary for transactivation by Ets factors with the
Tie2 promoter.13 The ability
of in vitro–translated human ELF-1 and cELF-1 to bind to the same Tie2
Ets sites was also examined. As shown in
Figure 8A, both human and chicken ELF-1 form similar
DNA-protein complexes with the Tie2 Ets sites (lanes 2 and 3). We then
tested the ability of an ELF-1–specific antibody to interfere with the
formation of these complexes. This antibody was able to interfere with
complex formation of both human and chicken ELF-1. Furthermore, the
appearance of an additional higher-mobility complex when the antibody
was used in the presence of cELF-1 suggests the formation of a
supershift (see arrow, lane 6). Because several Ets factors may
potentially bind to the Tie2 Ets site in vivo, in the developing
chicken, we performed gel-shift assays with the Tie2 Ets probe and cell
extracts from the chicken CAM. As shown in
Figure 8B, lane 3, a similar-sized complex is formed
compared with the in vitro–translated cELF-1. When the ELF-1 antibody
was added, it similarly resulted in the formation of a supershifted
complex, suggesting that cELF-1 derived from the chicken CAM is the Ets
factor that specifically binds to the Tie2 Ets site.
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We also examined the ability of cELF-1 to bind to
conserved Ets sites in the Tie1
promoter.9 There are 2
Ets-site doublets, P1 and P2, that are highly conserved in this
promoter. We tested the ability of cELF-1 to bind to these Ets sites.
As shown in
Figure 8C, cELF-1 binds well to both of these Ets sites. To
additionally demonstrate the specificity of binding to particular Ets
sites, we tested the ability of various cold mutant oligonucleotides to
interfere with binding of cELF-1 to the Tie1 P2 Ets sites
(Figure 8D). When both of the Ets sites were mutated (Mut 1,
2), the oligonucleotides were unable to compete for binding. When the
first Ets site was mutated, Mut 1, it competed only weakly with binding
of cELF-1 to the Tie1 P2 probe. However, when only the second Ets site
was mutated, it competed equally as well as the wild-type
oligonucleotide, suggesting that the first Ets site within this doublet
is a higher-affinity binding site for
cELF-1.
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The goal of this study was to identify transcription factors that regulate blood vessel development. In particular, we have identified a member of the Ets transcription-factor family, ELF-1, that is enriched in the developing blood vessels of the chicken embryo. ELF-1 was originally described as a regulator of T-cell–specific genes, including the interleukin-2 gene, interleukin-2 receptor, granulocyte-macrophage colony-stimulating factor, and CD4 genes.24 25 26 In addition, we and others have recently shown that ELF-1 is also expressed in B cells, where it regulates IgH gene expression.27 We have also recently isolated a novel member of the Ets gene family, NERF, which shares the highest degree of homology to ELF-1, and have shown that NERF and ELF-1 are also involved in the regulation of the B-cell–specific tyrosine kinase blk.23 This is the first report to demonstrate a role for ELF-1 in vascular-specific gene expression during blood vessel development in addition to its role in regulating genes of hematopoietic origin.
We have shown that ELF-1 can transactivate the Tie1 and Tie2 genes; therefore, these genes may serve as targets for ELF-1. The regulatory elements of both the Tie1 and Tie2 genes have been used to direct LacZ gene expression in a vascular-specific manner. Mutations in selected Ets sites in the regulatory regions of the Tie1 and Tie2 genes result in marked reductions in vascular-specific gene expression in vivo. Furthermore, with certain mutations, in addition to a reduction in overall vascular-specific gene expression, the LacZ-directed gene expression is reduced more in certain vascular beds than in others.12 This would suggest that certain Ets factors may be more important for the regulation of vascular-specific gene expression in certain vascular beds. With regard to expression of human and chicken ELF-1, for example, both are expressed only briefly in the fetal brain and not at all in adult brain, whereas both are strongly expressed in the fetal and adult heart, supporting a role for ELF-1 for differential gene expression in different tissues. A recent study performed on the transcriptional regulation of the human endothelial nitric oxide synthase (eNOS) gene provides additional support that ELF-1 is involved in vascular-specific gene expression. In this study, ELF-1 was shown to cooperate with the transcription factors Sp1, Sp3, and MAZ to form a multiprotein complex required for transcriptional activation of the eNOS gene.28
With regard to other Ets factors, Ets-1 has previously been shown to be expressed in developing blood vessels and tumor angiogenesis, but it does not seem to be able to transactivate the core regulatory elements of the Tie2 gene.29 30 Ets-1 may be important for normal endothelial function, because antisense oligonucleotides directed against Ets-1 were able to inhibit endothelial cell migration. Furthermore, these oligonucleotides were also able to inhibit vascular endothelial growth factor (VEGF)–induced cell proliferation.31 Ets-1 has also been shown to be inducible in endothelial cells by a variety of angiogenic substances, including basic fibroblast growth factor, VEGF, and epidermal growth factor. This upregulation was accompanied by increases in the expression of urokinase plasminogen activator and matrix metalloproteinase-1, which could be blocked with Ets-1 antisense oligonucleotides.32 However, targeted disruption of the gene was not associated with any vascular defects. One of the Ets factors, Tel, was recently shown to be involved in the development of the extraembryonic blood vessels. Targeted disruption of the gene leads to abnormalities in vitelline vein development. There seems to be normal capillary blood vessel development, but not of the larger blood vessels. The vascular defects seem to be mainly extraembryonic, with the predominant embryonic defects including apoptosis of mesenchymal and neural cells.33
Perhaps one of the most interesting recent findings regarding the transcriptional regulation of vascular development has been the determination that the zinc finger transcription factor stem-cell leukemia (SCL), which was originally shown to play a role strictly in hematopoiesis, also seems to be critical for embryonic blood vessel development. Targeted disruption of this gene led to embryonic lethality by day 9.5 because of an absence of yolk-sac erythropoiesis. However, it was unclear whether this gene might also contribute to nonhematopoietic pathways. By performing transgenic experiments in which the GATA-1 promoter was used to restore SCL gene expression in hematopoietic lineages in SCL-/- mice, there were striking abnormalities in yolk-sac angiogenesis.34 This suggests that certain transcription factors may be critical for both the normal development of hematopoietic cells and blood vessels and that there may be a common stem-cell precursor for both lineages. The most striking defects were a disorganized array of capillaries and absence of normal vitelline blood vessel formation. Although the larger vitelline blood vessels were not present, a smaller network of interconnecting vessels did exist. The architecture of these vessels revealed normal-appearing endothelial cells as well as the smooth muscle cells or pericytes that constituted the outer lining of the blood vessels.
In conclusion, the results of these studies provide substantial support for the role of the Ets factors in vascular development and vascular-specific gene expression in addition to their known role in hematopoiesis. The present study provides strong evidence for a dual role for ELF-1 in hematopoiesis and vascular development and, in particular, in regulating the gene expression of the Tie2 gene.
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Acknowledgments |
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This study was supported by National Institutes of Health grants RO1/HL63008 and KO8/CA71429 to P.O. and an American Heart Association Grant in Aid to L.I.
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Footnotes |
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Original received July 27, 2000; revision received November 2, 2000; accepted November 14, 2000.
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References |
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