|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 29 March 2007
doi: 10.1242/dev.001297
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Cell and Developmental Biology, University of Michigan Medical
School, Ann Arbor, MI 48109-2200, USA.
2 Department of Human Genetics, University of Michigan Medical School, Ann
Arbor, MI 48109-2200, USA.
3 TARA Centre, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577,
Japan.
Author for correspondence (e-mail:
engel{at}umich.edu)
Accepted 26 February 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Gata2, Endothelium, Cardiovascular, Lymphatic, Enhancer, ETS, SCL, Mouse
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Further
maturation of this new vasculature occurs via pruning of unneeded branches,
resulting in the formation of the mature vascular network.
In the yolk sac, the blood islands consist of a thin layer of angioblasts
surrounding primitive erythrocytes. Similarly, in the aorta-gonads-mesonephros
region - the initial embryonic site of definitive hematopoiesis -
hematopoietic stem cells can be detected budding from the endothelium of the
dorsal aorta (de Bruijn et al.,
2002). Given the close physical proximity of the very earliest
hematopoietic and endothelial cells, it has been speculated that they
originate from a common progenitor cell, which has been termed the
hemangioblast. A number of transcription factors have been shown to play a
role in the development of both cell lineages: for example, cloche is
required for the formation of endothelial and hematopoietic progenitors in
zebrafish (Stainier et al.,
1995) and Scl (also known as Tal1 - Mouse Genome
Informatics), which encodes a basic helix-loop-helix transcription factor, was
initially shown to be required for hematopoietic development in mice
(Robb et al., 1995;
Shivdasani et al., 1995).
Subsequent transgenic rescue of the hematopoietic defect in Scl-null
embryos revealed a requirement for SCL in the remodeling of the yolk sac
vasculature (Visvader et al.,
1998), and it has since been shown to play a role in
vasculogenesis (Patterson et al.,
2005), as well as in the migration and morphogenesis of
endothelial cells (Lazrak et al.,
2004). Transgenic expression of SCL is able to rescue the
phenotypic consequences of cloche mutation in the zebrafish,
suggesting that Scl functions downstream of cloche
(Liao et al., 1998). LMO2, a
member of the LIM domain family, is required for primitive erythropoiesis in
the embryo; Lmo2 ablation results in death at embryonic day (E) 9.75
secondary to hematopoietic failure (Warren
et al., 1994). Analysis of chimeric mice bearing contributions
from Lmo2-/- embryonic stem (ES) cells revealed that
angiogenic remodeling of blood vessels requires Lmo2
(Yamada et al., 2000).
Similarly, targeted disruption of the transcription factor Runx1
eliminates definitive hematopoiesis and results in defective angiogenesis and
hemorrhaging throughout the CNS (Wang et
al., 1996).
The most-widely accepted and experimentally supported model for lymphatic
development has proposed that the lymphatic vasculature arises from the blood
vasculature (Sabin, 1902;
Sabin, 1904;
Wigle and Oliver, 1999).
Expression of the lymphatic endothelial hyaluronan receptor gene
(Lyve1; also known as Xlkd1 - Mouse Genome Informatics) at
E9-9.5 in endothelial cells lining the anterior cardinal vein is the first
sign that these cells are competent to become lymphatic endothelial cells
(LECs). The lymphatic regulatory gene Prox1, encoding a homeobox
transcription factor, is expressed several hours later in a subset of
LYVE1+ cells in the anterior cardinal vein
(Oliver, 2004). Expression of
the murine vascular endothelial growth factor receptor 3 gene
(Vegfr3, also known as Flt4 - Mouse Genome Informatics),
which binds VEGFC, is detected in blood and lymphatic vessels during early
embryogenesis, but becomes largely restricted to lymphatic vessels after E14.5
(Kaipainen et al., 1995).
Beginning at E10.5, LECs bud and migrate away from the anterior cardinal
vein in a polarized non-random manner, and eventually fuse to form primitive
lymph sacs from which new LECs sprout and spread into the surrounding tissues
and organs (Wigle and Oliver,
1999). Finally, the lymphatic plexus undergoes remodeling and
maturation in the terminal stages of lymphatic development
(Oliver, 2004;
Oliver and Alitalo, 2005;
Sabin, 1902;
Sabin, 1904). Little is known
about the molecular events leading to lymphatic development, but gene-ablation
studies in mice and the identification of human hereditary-lymphedema
causative genes indicate that Prox1, Vegfc, Vegfr3, Foxc2 and
Sox18 are requisites to the process
(Wigle et al., 2002;
Wigle and Oliver, 1999;
Fang et al., 2000;
Irrthum et al., 2003;
Karkkainen et al., 2000;
Karkkainen et al., 2004;
Petrova et al., 2004).
GATA factors belong to an evolutionarily conserved family of C4
zinc-finger transcription factors that play demonstrably crucial roles in
development. There are six GATA family members in vertebrates, which have
historically been subdivided into two subfamilies. GATA-1, GATA-2 and GATA-3
are all important in the development of different hematopoietic lineages -
erythroid, hematopoietic progenitor and T-lymphoid, respectively - among many
other activities (Pandolfi et al.,
1995; Pevny et al.,
1995; Tsai et al.,
1994). Similarly, GATA-4, GATA-5 and GATA-6 have been shown to be
involved in cardiac, genitourinary and multiple endodermal developmental
events (Molkentin, 2000;
Molkentin et al., 1997;
Molkentin et al., 2000;
Morrisey et al., 1998).
GATA-2 was originally cloned from a chicken reticulocyte cDNA library
(Yamamoto et al., 1990), and
was shown to be expressed in a wide variety of tissues, including
hematopoietic, neuronal and endothelial cells. Gata2-null mutant
embryos die at mid-gestation due to a block in primitive hematopoiesis
(Tsai et al., 1994). Further
examination of Gata2 gain-of-function and in vitro differentiation of
Gata2-/- ES cells showed that GATA-2 plays a pivotal role
in the proliferation of very early hematopoietic progenitors
(Briegel et al., 1993;
Kitajima et al., 2002;
Tsai and Orkin, 1997),
underscoring the conclusions from the initial loss-of-function
experiments.
Given that many genes involved in hematopoiesis also participate in
vascular development and that GATA-2 is strongly expressed in endothelial cell
lines, it was originally believed that loss of GATA-2 function would result in
vascular defects. Adding further to this expectation was early evidence that
many genes that appeared to be crucial for endothelial development and
function are regulated via GATA-binding sites
(Dorfman et al., 1992). For
example, GATA sites have been implicated in the regulation of the
endothelium-specific genes preproendothelin (immature form of EDN1)
(Dorfman et al., 1992;
Yamashita et al., 2001),
Pecam1 (Gumina et al.,
1997), Vegfr2 (Kappel
et al., 2000; Minami et al.,
2004), eNOS (also known as Nos3 - Mouse Genome
Informatics) (German et al.,
2000) and Icam2 (Cowan
et al., 1998). Mutation of a GATA-binding site in the
Vegfr2 endothelium-specific enhancer completely abolished its
activity in transgenic reporter assays, indicating that Vegfr2
expression is dependent on GATA activity in vivo
(Kappel et al., 2000).
Surprisingly, however, the analysis of Gata2-null embryos failed to
reveal any obvious defects in the vasculature at the time of their early
embryonic demise (
E10) (Tsai et al.,
1994), leaving the role for GATA-2 in endothelial function
undefined.
To begin to investigate the role of GATA-2 in endothelial function, we systematically examined GFP expression in the developing vasculature of Gata2-GFP knock-in embryos during embryogenesis. We found that GFP was expressed in cells lining arterial and venous vessels formed during vasculogenesis and angiogenesis, and that its expression continued postnatally. We also observed GFP expression in budding LECs during early lymphatic development, as well as in postnatal lymphatic vessels. We then functionally identified an endothelium-specific enhancer in Gata2 intron 4 that could regulate the expression of a cis-linked reporter transgene in cardiovascular and lymphatic endothelial cells. Additionally, we found, using site-specific mutagenesis, that the potency of the minimal endothelium-specific enhancer is crucially dependent on an E box (CANNTG) motif. By contrast, disruption of three ETS-binding sites quantitatively reduced, but did not abolish, enhancer activity. Prior experiments showed that SCL activation is required for elaboration of the vasculature, and we demonstrate that SCL-E12 (E12 is also known as TCFE2A - Mouse Genome Informatics) heterodimers bind with high affinity to this crucial enhancer E box in vitro. Altogether, these data implicate ETS family members and SCL as in vivo activators of endothelium-specific Gata2 transcription.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Embryos were harvested at the times indicated in the text
and figure legends, and processed for immunostaining as previously described
(Khandekar et al., 2004).
Reactivity to rabbit anti-GFP (1:1000; Molecular Probes), rat anti-PECAM
(1:200; Pharmingen), goat anti-VEGFR3 (1:20; R&D Systems), rabbit
anti-PROX1 (1:800; Covance) and rabbit anti-LYVE1 (1:400; Upstate) antibodies
was detected using the appropriate fluorochrome-conjugated secondary
antibodies, as indicated in the figure legends. Digital images were recorded
as previously described (Khandekar et al.,
2004).
For founder transgenic analyses, expression constructs were purified for
microinjection into fertilized ova as previously described
(Khandekar et al., 2004). At
the indicated times, embryos from foster mothers or a Gata2 YAC d16Z
transgenic line were harvested for X-gal staining and PCR genotyping as
previously described (Zhou et al.,
1998). Transgenic embryos were photographed as whole-mount or
cryosectioned specimens as described previously
(Zhou et al., 1998).
Expression-plasmid construction
For microinjection, plasmid GR22-lacZ was digested with different
restriction enzymes (see legend to Fig.
3) (Zhou et al.,
2000). Other Gata2 fragments examined here were cloned
3' to the herpes simplex virus (HSV) thymidine kinase (TK) gene promoter
in TKß (Clontech) to mimic their natural position in the Gata2
locus. To generate TKBXß, a 2.9 kbp BamHI-SalI fragment
from plasmid GR22 was first subcloned into pBluescript II (Stratagene) and
then excised with XbaI before re-cloning into XbaI-digested
TKß. To generate TKSXß, TKBXß was treated with
SpeI-SfiI and T4 DNA polymerase before self-religation. To
construct TKAAß, a 460 bp AlwNI-ApaI fragment was
excised from plasmid GR22 and treated with T4 DNA polymerase before being
cloned into TKß, which had been treated sequentially with XbaI
and with Klenow polymerase. For microinjection, TKANß was generated from
TKAAß by NcoI restriction-enzyme digestion. To delete the
internal AlwNI-ApaI fragment, a plasmid subclone containing
the 1.2 kbp SfiI-XbaI Gata2 intron 4 was treated
with AlwNI-ApaI and T4 polymerase before self-religation.
The resultant 0.8 kbp SfiI-XbaI fragment was cloned into
TKß to generate TKSX
AAß. To clone the vascular
endothelium-specific (VE) enhancer into TKß (thereby generating
TKVEß, Fig. 5), primers
Endocons(f) and Endocons(r) containing an engineered XbaI site
(5'-ggtctagaCCATGGAGTCACCTATACTGTG-3' and
5'-ggtctagaACTGAGTCGAGGTGGCTCTG-3', respectively) were used to
generate a 167 bp amplicon (defined by the arrows in
Fig. 4A,
Fig. 5A), which was verified by
sequencing.
To mutate the E box in the VE enhancer, oligonucleotide-based PCR
mutagenesis was performed to introduce mutations (from
5'-CATCTG-3' to 5'-CAcccG-3'; mutations are lowercase)
that had been shown to eliminate SCL binding in gel shift assays
(Kappel et al., 2000). Primers
EcSCLmut(f) (5'-CGGACAcccGCAGCCG-3') and Endocons(r) (shown above)
were used to generate a 3' fragment using GR22 plasmid as template in a
PCR reaction. Similarly, EcSCLmut(r) (5'-CGGCTGCgggTGTCCG-3'] and
Endocons(f) were used to generate a 5' fragment. The resultant amplicons
were gel-purified and pooled as templates in a PCR reaction using Endocons(f)
and Endocons(r) as primers. The gel-purified PCR products were sequenced to
verify incorporation of the mutation and were then digested with XbaI
prior to cloning into the XbaI site of TKß to generate
TKVEßmScl (Fig. 5).
|
) into
all three ETS-binding sites in the Gata2 VE enhancer. The primers
Ecmets1(f) (5'-CTCCTGCCGcgGTTTCCTAT-3'), Ecmets1(r)
(5'-ATAGGAAACcgCGGCAGGAG-3'), Ecmets2(f)
(5'-TTCCTATCCGcgCATCTGCAG-3'), Ecmets2(r)
(5'-CTGCAGATGcgCGGATAGGAA-3'), Ecmets3(f)
(5'-TGTTTCCGcgCCGGCAA-3') and Ecmets3(r)
(5'-TTGCCGGcgCGGAAACA-3') were used to mutagenize the VE enhancer,
as described above, and the enhancer was then sequenced to verify
incorporation of the desired mutations. Fragments containing mutations in
either the first two or all three ETS-binding motifs were digested with
XbaI and then cloned into the XbaI site of TKß to
generate TKVEßmEts1,2 (data not shown) or TKVEßmEts1,2,3
(Fig. 5), respectively.
Electrophoretic mobility shift assay
Nuclear extracts were prepared as described previously
(Tanimoto et al., 2000) from
293T human embryonic kidney cells that were either mock transfected or
transfected with EF-1
promoter-directed SCL cDNA alone or with a CMV
promoter-directed E12 expression plasmid. Either no extract or nuclear extract
(35 µg) was added to binding buffer containing 20 mM HEPES (pH 7.9), 1 mM
MgCl2, 0.5 mM DTT and 37.5 ng/µl poly(dI-dC) at 4°C.
Unlabeled oligonucleotides [20- or 200-fold molar excess; wild type
(5'-TCCGGACATCTGCAGCCGGT-3'; E box underlined) or
mutant (5'-TCCGGACAcccGCAGCCGGT-3'; E box underlined,
mutated nucleotides lowercase)] or antibodies
(Rodriguez et al., 2005) were
added as indicated in the legend to Fig.
6. After 1 hour of pre-incubation, 2 µl (2x105
cpm) of radiolabeled wild-type oligonucleotide probe was added to each sample
and incubated for an additional 30 minutes. All samples were fractionated by
electrophoresis on neutral 6% TBE/polyacrylamide gels. After electrophoresis,
the gels were dried and recorded using a PhosphorImager (Molecular
Dynamics).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Umetani et al.,
2001), we first investigated whether or not Gata2 was
expressed in all endothelial cells in vivo by analyzing GFP staining in the
vasculature of Gata2-GFP knock-in heterozygotes
(Suzuki et al., 2006). In
whole-mount E9.5 embryos, robust GFP fluorescence was detected in the dorsal
aorta, a vessel formed by vasculogenesis, as well as in the developing heart
(Fig. 1A). In transverse
cryosections of E10.5 embryos, GFP-immunopositive cells were seen lining the
intersomitic vessels (Fig. 1B,
arrowheads), the aortic sac and ventricular endocardium
(Fig. 1C), and the developing
neural tube (Fig. 1D), a site
of intense sprouting angiogenesis. These data showed that GATA-2 is abundantly
expressed in vessels formed during primary vasculogenesis and
angiogenesis.
|
To verify that the GFP expression was indeed endothelium-specific, we performed co-immunostaining with antibodies directed against GFP and PECAM, an endothelial cell-specific cell-adhesion molecule. When E10.5 embryonic cryosections were stained with an anti-GFP antibody and a CY3-conjugated secondary antibody, vascular structures in the tail bud region stained strongly (Fig. 1G,J). When the same sections were co-stained for the detection of PECAM antigen using Alexa Fluor 488-conjugated secondary antibody (Fig. 1H,K), the anti-GFP and anti-PECAM immunofluorescence were completely coincident (Fig. 1I,L), verifying that Gata2 is expressed quite specifically in vascular endothelial cells.
To investigate whether Gata2 is expressed in the lymphatic vasculature, serial transverse cryosections of an E11.5 Gata2+/GFP embryo were stained with anti-VEGFR3 (Fig. 1M) or anti-GFP (Fig. 1N) antibodies. GFP fluorescence was detected strongly in the cardinal vein and dorsal aorta, as well as in clustered cells lying near the anterior cardinal vein (Fig. 1N, arrowheads). However, VEGFR3 immunoreactivity was most pronounced in scattered cells lying near the cardinal vein, from where LECs initially sprout (Fig. 1M, arrowheads). To confirm further that the GFP+/VEGFR3+ cells were indeed LECs, a second section was co-stained for the LEC-specific markers VEGFR3 and PROX1 (Fig. 1O). Nuclear anti-PROX1 and cytoplasmic anti-VEGFR3 immunofluorescence co-localized in the same cell population, which was located near the anterior cardinal vein (Fig. 1O, arrowhead). Similarly, GFP-expressing cells were identified in the blood and lymphatic vasculatures of the intestine and the mesentery (Fig. 1Q), and the skin (data not shown) of a postnatal day 1 (P1) pup. Expression of Vegfr3 (Fig. 1P), which is restricted to LECs after E14.5, co-localized with a subset of GFP-positive cells (Fig. 1R, arrowhead). Similar results were obtained with PROX1 and LYVE1 immunostaining (data not shown).
We conclude that GATA-2 is a pan-endothelial marker that is expressed early in lymphatic, vascular and endocardial endothelial cells. In the blood vasculature, it is indiscriminately expressed in blood vessels that are formed during vasculogenesis and angiogenesis, as well as in the arterial and venous branches of the embryonic vascular system. In the cardiovascular and lymphatic systems, Gata2 expression persists postnatally.
A transgenic YAC recapitulates endogenous Gata2 expression in the vasculature
We previously reported that a 271 kbp Gata2 yeast artificial
chromosome (YAC) transgene, containing sequences from -198 to +73 kbp (with
respect to the translation initiation site) of the Gata2 locus was
capable of rescuing the hematopoetic failure that is the underlying cause of
the early embryonic lethality in homozygous Gata2 mutant embryos
(Khandekar et al., 2004;
Zhou et al., 1998). When we
re-examined the ß-galactosidase (ß-gal) staining in the developing
vasculature of E8.5 transgenic embryos bearing the same (d16) YAC, but tagged
with lacZ (Zhou et al.,
1998), X-gal staining was very prominent in the heart tube
(Fig. 2A) and, by E10.5, in the
aorta and the endocardium (Fig.
2B). By E12.5, lacZ expression was pronounced throughout
the vascular system of the yolk sac and in the embryo proper
(Fig. 2C-E), although the
staining in the umbilical vein appeared to be fainter than in the umbilical
artery (Fig. 2E, uv and ua,
respectively). This differential staining was reminiscent of the differential
GFP intensity of expression observed earlier in the umbilical vein and artery
of Gata2-GFP knock-in heterozygotes. Furthermore, YAC d18Z (-40 to
+73 kbp) - a smaller, 5'-deletion derivative of d16Z
(Zhou et al., 1998;
Zhou et al., 2000) - displayed
an identical vascular lacZ pattern in transgenic embryos (data not
shown). This led to the tentative conclusion that the regulatory element(s)
directing Gata2 endothelial expression lay within the boundaries of
these YACs.
Localization of a Gata2 endothelium-specific enhancer
While investigating Gata2 activity in the developing nervous
system previously, we generated the plasmid GR22-lacZ, which contains
20 kbp of the Gata2 genomic sequence (from -9 kbp to slightly beyond
exon 6, with respect to the translational start site) with a lacZ
reporter gene inserted in frame at the initiation codon in exon 2
(Zhou et al., 2000). Plasmid
GR22-lacZ was separately digested with different restriction enzymes
in order to test, by founder transgenic analysis, overlapping fragments for
the presence of an endothelium-specific enhancer. Both
XhoI-SalI and KpnI-KpnI fragments
reproduced the same endothelium-restricted activity in the majority of
lacZ transgene-positive embryos (9/10 and 5/5, respectively;
Fig. 3A,B and data not shown)
(Zhou et al., 2000). Most
instructively, GR22-lacZ KpnI-SfiI transgenics displaced a
complete loss of endothelial X-gal staining (0/7 embryos;
Fig. 3C). Hence, the
Gata2 endothelium-specific enhancer activity could be tentatively
localized to within a 1.8 kbp SfiI-SalI interval in the
Gata2 fourth intron.
|
We tested next a series of smaller constructs (Fig. 4A) to establish the boundaries of a minimum enhancer element required to achieve the Gata2 endothelium-specific expression pattern. Deletion of the 1.2 kbp SX fragment from only the 3' end (in TKSRß) or from both termini (in TKAAß) did not alter the cardiovascular endothelium-specific expression in E10.5 transgenic embryos (Fig. 4B,C). By contrast, deletion of the internal AlwNI-ApaI (AA) 460 bp fragment from the SX enhancer fragment eliminated all endothelial enhancer activity (0/15; Fig. 4A,D). Thus, the minimal endothelium-specific enhancer as defined by the AA restriction fragment is sufficient for endothelium-specific Gata2 enhancer activity. Anti-LYVE1 immunostaining of E10.5 TKAAß transgenic embryos demonstrated that the minimal endothelium-specific enhancer remained active in LECs (data not shown).
Identification of key regulatory motifs within the Gata2 minimal endothelium-specific enhancer
Regulatory elements are thought to diverge more slowly than sequences that
surround them (Loots et al.,
2000). Comparison of the mouse Gata2 460 bp
endothelium-specific sequence to that of the human sequence demonstrated that
a 355 bp region within the AA fragment displayed 92% sequence identity, as
well as a nearby 58 bp region that harbored 96% identity (data not shown).
This extreme degree of evolutionary sequence conservation strongly implies an
associated functional significance. Analysis of the 460 bp element using
Matinspector 2.2 (Quandt et al.,
1995), which uses the consensus transcription factor-binding
motifs from the TRANSFAC database, identified a number of candidate regulatory
molecules that might bind to this enhancer
(Fig. 5A). A closer examination
of this restriction fragment showed an unusual clustering of binding sites
within a central 167 bp core region (as delineated by the two convergent
arrows shown in Fig. 4A and
Fig. 5A). Interestingly, the
3' terminus of this region corresponded closely to the RsrII
site that was identified previously as defining the 3' functional
boundary of endothelium-specific activity
(Fig. 4A,B).
|
|
; Zhou et al.,
2005). Whether or not these serve as bona fide binding motifs for
any of these factors, and determination of the underlying mechanism via which
they contribute to endocardial endothelial development, will require further
investigation. To ascertain whether the 5'-most 155 bp fragment was sufficient for endocardium-specific reporter gene activation, we tested the AlwNI-NcoI fragment in the context of TKß (Fig. 4A), and found that, of 11 recovered transgenic embryos, none displayed endocardial X-gal staining despite exhibiting variably ectopic X-gal staining (Fig. 4A,E). We conclude that the 5'-most 155 bp of the endothelium-specific enhancer alone is incapable of independently directing Gata2 endocardium expression.
|
; Wang et al.,
1997), and have also been reported to play prominent roles in
endothelium-specific enhancer activity
(Gottgens et al., 2002;
Kappel et al., 2000). The
existence of three ETS-binding sites within the 167 bp Gata2 VE
enhancer suggested that one or multiple Ets family members might modulate its
activity. To directly address this hypothesis, the three ETS-binding sites
were individually mutated from CGGA to CGcg, a mutation that was previously
shown to eliminate ETS-factor binding
(O'Reilly et al., 2003).
Mutation of either two or all three ETS-binding sites reduced the overall
number of embryos displaying weak, albeit endothelium-specific, staining (1/12
and 3/25, respectively; data not shown and
Fig. 5C).
Next we determined whether the single E box motif present in the 167 bp VE
enhancer is important for its overall activity. A mutation
(5'-CATCTG-3' to 5'-CAcccG-3') that was previously
shown to eliminate SCL binding (Kappel et
al., 2000) was incorporated into the TKVEß plasmid. Among the
24 recovered TKVEßmScl transgenic embryos, none displayed endothelial
ß-gal staining (e.g. Fig.
5D). This lack of staining was presumably not due to the effect of
transgene integration site, because some of the transgenic embryos (9/24)
exhibited ectopic staining in the spinal cord or head. These data demonstrate
that the single Gata2 intron 4 E box exerts a profound effect on the
activity of the VE enhancer and, by extension, in the regulation of
Gata2 expression throughout the blood endothelium.
An SCL-E12 heterodimer avidly binds to the E box motif in the Gata2 VE enhancer
Because SCL had been shown previously to be essential for endothelial
differentiation (Visvader et al.,
1998), we wished to determine whether SCL could bind to the E box
in the VE fragment. To do so, we performed electrophoretic mobility shift
assays (EMSAs) using nuclear extracts from 293T cells transfected with either
SCL, or SCL plus E12, expression vectors. Incubation of radiolabeled E box
oligonucleotide probes with nuclear extracts containing SCL-E12, but not SCL
alone, resulted in a low-mobility complex
(Fig. 6, lane 4), which could
be specifically competed by the addition of an excess of unlabeled E box
oligonucleotide (Fig. 6, lanes
5,6), but not by mutant E box oligonucleotide
(Fig. 6, lanes 7,8). The
binding specificity and protein identity were confirmed by showing that
addition of an anti-SCL antibody (Fig.
6, lanes 9,10), but not control IgG
(Fig. 6, lane 11),
significantly reduced EMSA complex formation. Thus, the crucial E box motif in
the Gata2 VE enhancer can be strongly bound by the basic
helix-loop-helix transcription factor SCL.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Identification of Gata2 pan-endothelium-specific enhancer
The existence of an endothelial enhancer for Gata2 raises several
intriguing questions about its function. Earlier genetic data indicated that
GATA-2 plays a crucial role in early hematopoietic development
(Tsai et al., 1994) and is
indeed capable of specifically marking hematopoietic stem cells
(Suzuki et al., 2006). Given
that the earliest hemangiogenic cells are closely related to the endothelial
lineage (Choi et al., 1998),
one might speculate that this endothelial enhancer could also target a subset
of hematopoietic cells that are generated from the aortic endothelium.
Suggestively, we identified lacZ-positive cells that appear to be
`budding' from the aortic wall (Fig.
3E, arrow). Whether these cells are bona fide hematopoietic cells
or simply endothelial cells being sloughed into the aorta is yet to be
determined. However, the existence of an endothelium-specific enhancer of
Scl, which also marks hematopoietic progenitors
(Gottgens et al., 2004),
suggests that the Gata2 intron 4 enhancer identified here may play
some role in the appropriate regulation of Gata2 in hematopoietic
progenitors as well, particularly in light of the recent observation that this
enhancer is active in definitive erythroid cells
(Grass et al., 2006) (see
below).
We report here that Gata2 is also expressed at the earliest stage of lymphangiogenesis, when LECs bud from the anterior cardinal veins at mid-gestation, and that it continues to be expressed in the postnatal lymphatic vasculature. Although the initial budding of PROX1+ LECs appeared to be normal in GATA-2-deficient embryos (data not shown), it remains to be determined whether lymphatic development after E10, the nominal time of death of Gata2-null embryos, continues unperturbed.
In the blood vasculature, the lack of any reported phenotype in
Gata2-/- embryos led to the initial conclusion that
functional redundancy of other GATA family members, including GATA-4 and
GATA-6, may compensate for any lack of GATA-2 in the endothelium. Another
equally plausible possibility is that Gata2 mutant embryos simply die
too early (E10) (Tsai et al.,
1994) to generate a robust vascular phenotype. Based on the
analysis of Gata3-lacZ knock-in mice, Gata3 expression in
the endothelium does not appear to be widespread, but this closely related
GATA family member does seem to be sporadically active (our unpublished data).
Gata4, which plays a crucial role in heart development, does not
appear to be expressed in mature endothelial cells
(Umetani et al., 2001),
although it is expressed in endothelial progenitors
(Hatzopoulos et al., 1998).
Gata6 is expressed in both endothelial precursors as well as in
mature endothelial cells (Hatzopoulos et
al., 1998; Umetani et al.,
2001), making GATA-6 a prime candidate for a possible
Gata2-complementing endothelium activity. Whereas Gata4 and
Gata6 heterozygotes are normal, compound heterozygotes display
cardiovascular defects (Xin et al.,
2006). Notably, these embryos displayed a less intricate weave,
which was disorganized, of the cranial and intersomitic vasculature, as well
as hemorrhaging. The recent generation of a Gata6 conditional
loss-of-function allele (Sodhi et al.,
2006) should now permit exploration of cell autonomous GATA-6
involvement, if any, in endothelial development.
Role of Ets family transcription factors in Gata2 endothelium-specific enhancer activity
Within an initial functionally defined restriction fragment describing the
Gata2 endothelial enhancer, we subsequently identified a 167 bp core
enhancer that was sufficient to recapitulate vascular endothelial expression.
The existence of three putative Ets family-member-binding sites implicated a
role for these factors in the control of this VE enhancer. The Ets family of
transcription factors have been shown to play an important role in vascular
development in vivo (Sumanas and Lin,
2006) and have been shown to be functionally important in the
activation of a number of endothelial-specific enhancers, including
Scl (Gottgens et al.,
2004), Tie2 (also known as Tek)
(Minami et al., 2003) and
Flk1 (also known as Kdr)
(Elvert et al., 2003).
Furthermore, disruption of Tel (also known as Etv6), one Ets
family member, results in defective yolk sac angiogenesis
(Wang et al., 1997),
suggesting that TEL plays an important role in vascular remodeling. However,
targeted mutation of other ETS factors has not revealed vascular deficiencies,
suggesting that these factors may either play no role or may also be
functionally redundant in endothelium development.
In the Gata2 VE enhancer defined here, mutations predicted to
disrupt ETS-binding sites significantly attenuated enhancer activity, as
indicated by the number of, and X-gal-staining intensity in, transgenic
embryos displaying endothelial ß-gal staining. However, the weak staining
pattern detected in these embryos appeared to remain endothelium-specific,
indicating that the ETS-binding sites are not essential for the tissue
specificity of the enhancer, but rather may serve to augment its overall
potency. Because this cis mutation has been shown in a similar assay to
eliminate DNA binding for some members of the family
(O'Reilly et al., 2003), we
cannot rule out the possibility that the mutation does not abolish the binding
of the multiple Ets family members that are expressed within the endothelium
(Lelievre et al., 2001).
Additionally, the heterogeneity of ETS-binding sites suggests that some family
members may be able to bind to other sequences within the enhancer, enabling
endothelial activation despite mutations within canonical high-affinity
binding sites.
An E box-binding factor is required for Gata2 endothelial enhancer activity
The transcription factor SCL has been shown to play crucial roles in both
hematopoiesis (Shivdasani et al.,
1995) and vascular development
(Patterson et al., 2005;
Visvader et al., 1998),
leading to the speculation that SCL may be important for the ontogeny of the
hemangioblast. A comprehensive analysis of the transcriptional regulation of
Scl has identified several tissue-specific enhancers that are
required for its appropriate expression
(Barton et al., 2001;
Gottgens et al., 2002;
Sinclair et al., 1999).
Interestingly, the enhancer specific for hematopoietic progenitors has GATA
sites that are crucial for Scl enhancer activity in vivo. The factor
responsible for binding to these sites in hematopoietic cell lines appears to
be GATA-2, suggesting that GATA-2 is responsible for activating Scl
in early hematopoiesis. However, there is no GATA-binding site in the
endothelial enhancer of human SCL, although it remains possible that
a GATA factor is acting without directly binding to DNA
(Gottgens et al., 2004). The
data presented here are consistent with the possibility that Gata2
and Scl encode reciprocally reinforcing activators in these
developmentally related tissues, although other interpretations are clearly
not excluded from the data presented.
To assess the relationship between Scl and Gata2 in the
endothelium, we mutated the single E box present within the 167 bp
Gata2 VE enhancer. Scl has previously been shown to be
regulated by GATA factors in both the CNS
(Sinclair et al., 1999) and
hematopoietic progenitors (Gottgens et
al., 2002), suggesting that the nature of the epistatic
relationship between these two factors may be dependent on the specific tissue
in question. However, these data also underscore the point that the functions
of these two factors are often intimately intertwined during development.
Lending further credence to this point is the evidence that Scl
expression in the endothelium is crucially dependent on Ets-family activity
(Gottgens et al., 2004). The
endothelial enhancer of Scl contains five ETS-binding sites that are
required for the activity of the enhancer in trans-activation assays.
Given that both Scl and Gata2 appear to be regulated by ETS
factors in the endothelium, we surmise that ETS, SCL and GATA-2 together
constitute a regulatory circuitry wherein, in the simplest scenario envisaged,
ETS factors activate Scl, and ETS and SCL then cooperate to activate
Gata2 in endothelial cells. Similarly, the data are also consistent
with the possibility that Ets family members collaborate with GATA-2 to
reinforce Scl expression in a positive-feedback loop.
The similarities between the regulation of Scl and Gata2
are also underscored by the similarity of their functions in the hematopoietic
system. Targeted mutation of both genes results in defects in both primitive
and definitive hematopoiesis, resulting in mid-gestational lethality
(Shivdasani et al., 1995;
Tsai et al., 1994). This
phenotypic similarity has not been demonstrated in the vascular system, where
SCL has been shown to play a prominent role in vascular remodeling, whereas
GATA-2 has not. However, because the vascular defects in Scl-null
mutants were only revealed after selective rescue of hematopoiesis, it seems
likely that the early lethality of Gata2-null mice precludes a more
precise analysis of the function of GATA-2 in the vascular system. Experiments
are underway to circumvent the embryonic hematopoietic lethality and explore
possible functions of GATA-2 in the vasculature.
The presence of multiple GATA-binding motifs within the VE enhancer also
raises the issue of whether this enhancer might be auto-regulated by GATA-2,
or even by another GATA factor. Of specific interest here, Grass et al.
recently identified this same element through its evolutionary sequence
conservation during an analysis of GATA-1 regulation of the Gata2
gene in erythroid cells (Grass et al.,
2006). In that study, the authors demonstrated that sequences
overlapping the VE element exhibited robust activity in transfected erythroid
cells and that elimination of the GATA sites abrogated the erythroid enhancer
activity. Here, we show that the VE element is at least equally as active and
as specific for endothelial cells in a rigorous in vivo assay. Whether the
activity identified by Grass et al. and the activity we defined here
represents an endothelial-enhancer activity that can simply be surreptitiously
activated in erythroid cells or whether the element represents one that can be
a bona fide target for GATA-factor activation in both hematopoietic and
endothelial cells (as one might imagine for a hemangioblast-responsive
element) awaits resolution following further investigation.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Aveo Pharmaceuticals, 75 Sidney Street, Cambridge, MA
02139, USA
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ayadi, A., Zheng, H., Sobieszczuk, P., Buchwalter, G., Moerman, P., Alitalo, K. and Wasylyk, B. (2001). Net-targeted mutant mice develop a vascular phenotype and up-regulate egr-1. EMBO J. 20,5139 -5152.[CrossRef][Medline]
Barton, L. M., Gottgens, B., Gering, M., Gilbert, J. G.,
Grafham, D., Rogers, J., Bentley, D., Patient, R. and Green, A. R.
(2001). Regulation of the stem cell leukemia (SCL) gene: a tale
of two fishes. Proc. Natl. Acad. Sci. USA
98,6747
-6752.
Briegel, K., Lim, K. C., Plank, C., Beug, H., Engel, J. D. and
Zenke, M. (1993). Ectopic expression of a conditional
GATA-2/estrogen receptor chimera arrests erythroid differentiation in a
hormone-dependent manner. Genes Dev.
7,1097
-1109.
Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, J. C. and Keller, G. (1998). A common precursor for hematopoietic and endothelial cells. Development 125,725 -732.[Abstract]
Cowan, P. J., Tsang, D., Pedic, C. M., Abbott, L. R., Shinkel,
T. A., d'Apice, A. J. and Pearse, M. J. (1998). The human
ICAM-2 promoter is endothelial cell-specific in vitro and in vivo and contains
critical Sp1 and GATA binding sites. J. Biol. Chem.
273,11737
-11744.
de Bruijn, M. F., Ma, X., Robin, C., Ottersbach, K., Sanchez, M. J. and Dzierzak, E. (2002). Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta. Immunity 16,673 -683.[CrossRef][Medline]
Dorfman, D. M., Wilson, D. B., Bruns, G. A. and Orkin, S. H.
(1992). Human transcription factor GATA-2. Evidence for
regulation of preproendothelin-1 gene expression in endothelial cells.
J. Biol. Chem. 267,1279
-1285.
Elvert, G., Kappel, A., Heidenreich, R., Englmeier, U., Lanz,
S., Acker, T., Rauter, M., Plate, K., Sieweke, M., Breier, G. et al.
(2003). Cooperative interaction of hypoxia-inducible
factor-2alpha (HIF-2alpha) and Ets-1 in the transcriptional activation of
vascular endothelial growth factor receptor-2 (Flk-1). J. Biol.
Chem. 278,7520
-7530.
Fang, J., Dagenais, S. L., Erickson, R. P., Arlt, M. F., Glynn, M. W., Gorski, J. L., Seaver, L. H. and Glover, T. W. (2000). Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedema-distichiasis syndrome. Am. J. Hum. Genet. 67,1382 -1388.[CrossRef][Medline]
German, Z., Chambliss, K. L., Pace, M. C., Arnet, U. A.,
Lowenstein, C. J. and Shaul, P. W. (2000). Molecular basis of
cell-specific endothelial nitric-oxide synthase expression in airway
epithelium. J. Biol. Chem.
275,8183
-8189.
Gottgens, B., Nastos, A., Kinston, S., Piltz, S., Delabesse, E. C., Stanley, M., Sanchez, M. J., Ciau-Uitz, A., Patient, R. and Green, A. R. (2002). Establishing the transcriptional programme for blood: the SCL stem cell enhancer is regulated by a multiprotein complex containing Ets and GATA factors. EMBO J. 21,3039 -3050.[CrossRef][Medline]
Gottgens, B., Broccardo, C., Sanchez, M. J., Deveaux, S.,
Murphy, G., Gothert, J. R., Kotsopoulou, E., Kinston, S., Delaney, L., Piltz,
S. et al. (2004). The scl +18/19 stem cell enhancer is not
required for hematopoiesis: identification of a 5' bifunctional
hematopoietic-endothelial enhancer bound by Fli-1 and Elf-1. Mol.
Cell. Biol. 24,1870
-1883.
Grass, J. A., Jing, H., Kim, S. I., Martowicz, M. L., Pal, S.,
Blobel, G. A. and Bresnick, E. H. (2006). Distinct functions
of dispersed GATA factor complexes at an endogenous gene locus.
Mol. Cell. Biol. 26,7056
-7067.
Gumina, R. J., Kirschbaum, N. E., Piotrowski, K. and Newman, P.
J. (1997). Characterization of the human platelet/endothelial
cell adhesion molecule-1 promoter: identification of a GATA-2 binding element
required for optimal transcriptional activity. Blood
89,1260
-1269.
Hatzopoulos, A. K., Folkman, J., Vasile, E., Eiselen, G. K. and Rosenberg, R. D. (1998). Isolation and characterization of endothelial progenitor cells from mouse embryos. Development 125,1457 -1468.[Abstract]
Irrthum, A., Devriendt, K., Chitayat, D., Matthijs, G., Glade, C., Steijlen, P. M., Fryns, J. P., Van Steensel, M. A. and Vikkula, M. (2003). Mutations in the transcription factor gene SOX18 underlie recessive and dominant forms of hypotrichosis-lymphedema-telangiectasia. Am. J. Hum. Genet. 72,1470 -1478.[CrossRef][Medline]
Kaipainen, A., Korhonen, J., Mustonen, T., van Hinsbergh, V. W.,
Fang, G. H., Dumont, D., Breitman, M. and Alitalo, K. (1995).
Expression of the fms-like tyrosine kinase 4 gene becomes restricted to
lymphatic endothelium during development. Proc. Natl. Acad. Sci.
USA 92,3566
-3570.
Kappel, A., Schlaeger, T. M., Flamme, I., Orkin, S. H., Risau,
W. and Breier, G. (2000). Role of SCL/Tal-1, GATA, and ets
transcription factor binding sites for the regulation of flk-1 expression
during murine vascular development. Blood
96,3078
-3085.
Karkkainen, M. J., Ferrell, R. E., Lawrence, E. C., Kimak, M. A., Levinson, K. L., McTigue, M. A., Alitalo, K. and Finegold, D. N. (2000). Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nat. Genet. 25,153 -159.[CrossRef][Medline]
Karkkainen, M. J., Haiko, P., Sainio, K., Partanen, J., Taipale, J., Petrova, T. V., Jeltsch, M., Jackson, D. G., Talikka, M., Rauvala, H. et al. (2004). Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat. Immunol. 5,74 -80.[CrossRef][Medline]
Khandekar, M., Suzuki, N., Lewton, J., Yamamoto, M. and Engel,
J. D. (2004). Multiple, distant Gata2 enhancers specify
temporally and tissue-specific patterning in the developing urogenital system.
Mol. Cell. Biol. 24,10263
-10276.
Kitajima, K., Masuhara, M., Era, T., Enver, T. and Nakano, T. (2002). GATA-2 and GATA-2/ER display opposing activities in the development and differentiation of blood progenitors. EMBO J. 21,3060 -3069.[CrossRef][Medline]
Lazrak, M., Deleuze, V., Noel, D., Haouzi, D., Chalhoub, E.,
Dohet, C., Robbins, I. and Mathieu, D. (2004). The bHLH
TAL-1/SCL regulates endothelial cell migration and morphogenesis.
J. Cell Sci. 117,1161
-1171.
Lelievre, E., Lionneton, F., Soncin, F. and Vandenbunder, B. (2001). The Ets family contains transcriptional activators and repressors involved in angiogenesis. Int. J. Biochem. Cell Biol. 33,391 -407.[CrossRef][Medline]
Liao, E. C., Paw, B. H., Oates, A. C., Pratt, S. J.,
Postlethwait, J. H. and Zon, L. I. (1998). SCL/Tal-1
transcription factor acts downstream of cloche to specify hematopoietic and
vascular progenitors in zebrafish. Genes Dev.
12,621
-626.
Loots, G. G., Locksley, R. M., Blankespoor, C. M., Wang, Z. E.,
Miller, W., Rubin, E. M. and Frazer, K. A. (2000).
Identification of a coordinate regulator of interleukins 4, 13,
and 5 by cross-species sequence comparisons. Science
288,136
-140.
Minami, T., Kuivenhoven, J. A., Evans, V., Kodama, T.,
Rosenberg, R. D. and Aird, W. C. (2003). Ets motifs are
necessary for endothelial cell-specific expression of a 723-bp Tie-2
promoter/enhancer in Hprt targeted transgenic mice. Arterioscler.
Thromb. Vasc. Biol. 23,2041
-2047.
Minami, T., Murakami, T., Horiuchi, K., Miura, M., Noguchi, T.,
Miyazaki, J., Hamakubo, T., Aird, W. C. and Kodama, T.
(2004). Interaction between hex and GATA transcription factors in
vascular endothelial cells inhibits flk-1/KDR-mediated vascular endothelial
growth factor signaling. J. Biol. Chem.
279,20626
-20635.
Molkentin, J. D. (2000). The zinc
finger-containing transcription factors GATA-4, -5, and -6. Ubiquitously
expressed regulators of tissue-specific gene expression. J. Biol.
Chem. 275,38949
-38952.
Molkentin, J. D., Lin, Q., Duncan, S. A. and Olson, E. N.
(1997). Requirement of the transcription factor GATA4 for heart
tube formation and ventral morphogenesis. Genes Dev.
11,1061
-1072.
Molkentin, J. D., Tymitz, K. M., Richardson, J. A. and Olson, E.
N. (2000). Abnormalities of the genitourinary tract in female
mice lacking GATA5. Mol. Cell. Biol.
20,5256
-5260.
Morrisey, E. E., Tang, Z., Sigrist, K., Lu, M. M., Jiang, F.,
Ip, H. S. and Parmacek, M. S. (1998). GATA6 regulates HNF4
and is required for differentiation of visceral endoderm in the mouse embryo.
Genes Dev. 12,3579
-3590.
Nemer, G. and Nemer, M. (2002). Cooperative
interaction between GATA5 and NF-ATc regulates endothelial-endocardial
differentiation of cardiogenic cells. Development
129,4045
-4055.
O'Reilly, D., Quinn, C. M., El-Shanawany, T., Gordon, S. and
Greaves, D. R. (2003). Multiple Ets factors and interferon
regulatory factor-4 modulate CD68 expression in a cell type-specific manner.
J. Biol. Chem. 278,21909
-21919.
Oliver, G. (2004). Lymphatic vasculature development. Nat. Rev. Immunol. 4, 35-45.[CrossRef][Medline]
Oliver, G. and Alitalo, K. (2005). The lymphatic vasculature: recent progress and paradigms. Annu. Rev. Cell Dev. Biol. 21,457 -483.[CrossRef][Medline]
Pandolfi, P. P., Roth, M. E., Karis, A., Leonard, M. W., Dzierzak, E., Grosveld, F. G., Engel, J. D. and Lindenbaum, M. H. (1995). Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis. Nat. Genet. 11,40 -44.[CrossRef][Medline]
Patterson, L. J., Gering, M. and Patient, R.
(2005). Scl is required for dorsal aorta as well as blood
formation in zebrafish embryos. Blood
105,3502
-3511.
Petrova, T. V., Karpanen, T., Norrmen, C., Mellor, R., Tamakoshi, T., Finegold, D., Ferrell, R., Kerjaschki, D., Mortimer, P., Yla-Herttuala, S. et al. (2004). Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis. Nat. Med. 10,974 -981.[CrossRef][Medline]
Pevny, L., Lin, C. S., D'Agati, V., Simon, M. C., Orkin, S. H. and Costantini, F. (1995). Development of hematopoietic cells lacking transcription factor GATA-1. Development 121,163 -172.[Abstract]
Quandt, K., Frech, K., Karas, H., Wingender, E. and Werner, T. (1995). MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide se
