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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4196-4207
The Mouse GATA-2 Gene is Expressed in the Para-Aortic
Splanchnopleura and Aorta-Gonads and Mesonephros Region
By
Naoko Minegishi,
Jun Ohta,
Hironori Yamagiwa,
Norio Suzuki,
Shimako Kawauchi,
Yinghui Zhou,
Satoru Takahashi,
Norio Hayashi,
James Douglas Engel, and
Masayuki Yamamoto
From the Center for Tsukuba Advanced Research Alliance and Institute
of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan;
Department of Biochemistry, Tohoku University School of Medicine, 2-1 Seiryocho, Aoba-ku, Sendai, Japan; and Department of Biochemistry,
Molecular Biology and Cell Biology, Northwestern University, Evanston
IL.
 |
ABSTRACT |
We previously reported that the mouse GATA-2 gene is
regulated by two alternative promoters (Minegishi et al, J
Biol Chem, 273:3625, 1998). Although the more proximal IG (general)
promoter is active in almost all GATA-2-expressing cells, the distal
IS (specific) promoter activity was selectively detected in
hematopoietic tissues but not in other mesodermal tissues. We report
here in vivo analysis of the GATA-2 locus and its regulatory
characteristics in hematopoietic tissues of transgenic mice. Transgenes
containing 6 or 7 kbp of sequence flanking the 5' end of the IS
first exon direct expression of  -galactosidase or green fluorescent
protein (GFP) reporter genes specifically to the para-aortic
splanchnopleura, aorta-gonads, and mesonephros (AGM) region, and in the
neural tissues. In situ hybridization analysis showed that reporter
gene expression specifically recapitulates the endogenous expression profile of GATA-2 in these tissues. The flk-1, CD34, c-kit, and CD45
antigens were identified in the GFP-positive cells from the AGM region
and fetal liver, indicating that GATA-2 is expressed in immature
hematopoietic cells. Deletion of 3.5 kbp from the 5' end of the
6.0 kbp IS promoter construct, including one of the DNase I
hypersensitive sites, completely abolished hematopoietic expression.
These experiments describe an early developmental GATA-2 hematopoietic
enhancer located between 6.0 and 2.5 kbp 5' to the IS exon.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIESIS IS REGULATED, in part, by a
network of lineage-restricted as well as ubiquitous transcription
factors. In particular, the GATA transcription factor family has been
shown to play prominent regulatory roles in multiple hematopoietic
lineages.1,2 The founding member of the family, GATA-1, is
essential for the terminal differentiation of erythroid cells,
megakaryocytes, mast cells, and eosinophils.3-8 GATA-1 is
also expressed from an alternative promoter and first exon in Sertoli
cells of the testis.9-11 Acting at an earlier step than
GATA-1, GATA-2 has been shown to be essential for early involvement in
the regulatory network controlling hematopoietic progenitors.12 In gene targeting experiments, the loss of
GATA-2 in mice was shown to confer embryonic lethality with severe
anemia at 10-days postcoitus (dpc), and the affected embryos manifested a broad hematopoietic deficit.12 In vitro differentiation
of GATA-2 (-/-) ES cells showed that GATA-2 is required for the
proliferation and/or survival of definitive hematopoietic progenitors
and mast cells.12
The expression of GATA-2 gene is common in hematopoietic
lineages in all vertebrates.13-19 GATA-2 was originally
cloned from a chicken reticulocyte complementary DNA (cDNA)
library,13 and has been shown to be expressed in a wide
variety of tissues (eg, endothelial cells, brain, sympathetic neurons,
fibroblasts, kidney, liver, ovary, lung, and cardiac
muscle).13,18,20 Within hematopoietic cell lineages, GATA-2
is expressed in the stem and/or progenitor cell
fraction,14,21-23 as well as in immature erythroid cells, mast cells, eosinophils, and megakaryocytes.23-27 In
Xenopus, GATA-2 is known to be expressed as a maternal messenger RNA
(mRNA) in oocytes, and zygotic expression of GATA-2 mRNA starts in the
ventral mesoderm, the initial site of hematopoiesis in the
frog.15,16,28,29 This restricted expression of the
GATA-2 gene is under the positive control of BMP-4 and the
negative regulatory influence of activin and noggin.15,30
In zebrafish, GATA-2 is expressed in the yolk syncytial layer and the
intermediate cell mass, both of which contain hematopoietic
progenitors.17,19 These data strongly imply GATA-2 as an
important factor in the ontogeny of hematopoietic cell development.
We have recently found that the mouse GATA-2
(mGATA-2) gene is regulated by two
promoters.31 Reverse transcriptase-polymerase chain
reaction (RT-PCR) analysis showed that the proximal (IG) promoter, a
counterpart to the previously reported Xenopus32 and human33 GATA-2 promoters, was generally used in adult
tissues that express GATA-2 protein.31 In contrast, the
distal (IS) promoter was activated specifically in hematopoietic but
not in other mesodermal tissues.31 When examined in
transient transfection assays, both the IS and IG promoters were
regulated by regions within 100 bp from their respective transcription
start sites.31 When more extensive regions of the locus
were examined, no additional transcriptional activities were
shown.31 Thus the data suggested that activities determined
using the transient transfection assay might be somewhat different from
those that must be operative in vivo.
To understand the regulatory mechanisms controlling GATA-2 gene
expression in hematopoietic stem cells and progenitors, we set
out to examine the regulatory activity of the mGATA-2 genomic locus in
vivo using transgenic mice. Here we focused on the role of the GATA-2
IS promoter in early hematopoietic development. The results show that a
discrete hematopoietic element, lying between 2.5 and 6.0 kbp 5'
to the IS promoter, directs GATA-2 gene expression in the
para-aortic splanchnopleura and aorta, gonads, and mesonephros (AGM)
region, in which definitive hematopoiesis is believed to
commence.34, 35
| |
MATERIALS AND METHODS |
In situ hybridization analysis.
Digoxygenin (DIG)-labeled riboprobes used for both the whole-mount and
tissue section in situ hybridization analyses were prepared following
the manufacturer's recommendations (Boehringer Mannheim, Mannheim,
Germany). Antisense and sense probes were generated from a
Bluescript plasmid (Stratagene, La Jolla, CA) containing a
partial mGATA-2 cDNA (nt 170 to 871)36 using T3 or T7 RNA
polymerase, respectively. To reduce nonspecific binding, these probes
did not contain the zinc finger region of mGATA-2, which is highly
conserved among the GATA factors.
Mapping of DNase I hypersensitive sites.
Nuclei were isolated from P815 mastocytoma cells (generously provided
by Dr Atushi Ichikawa, Kyoto University, Kyoto, Japan) and digested
with serial dilution of DNase I. DNA was extracted from the treated
nuclei and Southern blot hybridization was performed as described
previously.37 Probes used were a PstI-SacI
fragment (probe a; 0.6 kbp) and a NotI-BamHI
fragment (probe b; 0.6 kbp), both of which contain the IS exon.
mGATA-2 transgene constructs.
Various mGATA-2 genomic fragments lying 5' to the
NotI site in the IS exon were ligated into the XhoI
site of pSV (Clontech, Palo Alto, CA) containing
-galactosidase (LacZ) gene. These plasmids, designated p6.0ISLacZ,
p2.5ISLacZ, and p0.65ISLacZ, were injected into fertilized murine eggs.
A genomic promoter fragment extending from a HindIII site (7.0 kbp 5' to the IS exon) to the NotI site, as well as a DNA
fragment containing the splice donor and acceptor sequences of pSV,
were ligated to the green fluorescent protein (GFP) cDNA in
pCMX-SAH/Y145F plasmid (the kind gift from Dr Kazuhiko Umesono, Kyoto
University). A SacI-EcoRV fragment from this plasmid intermediate was then introduced into the SacI and NotI
sites of pSV. This final construct, referred to as p7.0ISGFP,
additionally contained the polyadenylation consensus sequence of
pSV.
Transgenic mice.
Linearized plasmids were purified by NACS PREPAC (GIBCO
BRL, Rockville, MD), adjusted to 5 ng/mL and injected into
mouse oocytes as described.10 Transgene integration was
confirmed by PCR of genomic DNA from mouse tails. The primers used
were: IS primer, 5'-ACAAAAGCGGCTGTCTGCGCGACG-3'; IG primer,
5'-CACCCCTATCCCGTGAATCCG-3'; LacZ primer,
5'-GCAACGAAAATCACGTTCTTGT-3'; GFP primer,
5'-TGCCGTCGTCCTTGAAGAAGATG-3'. The transgenes copy numbers
were estimated by Southern blot analysis.10 LacZ staining
and RT-PCR analysis were performed as previously described.31,38 Fluorescence-activated cell sorting (FACS) was performed as previously described,31,38 using FACS
Vantage and FACS Caliber equipment (Becton Dickinson, Mountain View,
CA). Anti-flk-1 antibody (Avas12)39 was
kindly provided by Dr Shin-ichi Nishikawa (Kyoto University), and was
biotinylated using EZ-link NHS-LC-Biotin (PIERCE, Rockford,
IL). Phycoerythrin (PE)-conjugated anti-c-kit, anti-Sca1
and anti-CD45 antibodies, biotin-conjugated anti-CD34 and anti-Ter119
antibodies, and streptavidin allophycocyanine (APC) were purchased from
PharMingen (San Diego, CA).
| |
RESULTS |
Expression of GATA-2 in hematopoietic tissues.
To begin to elucidate the mechanisms that regulate mGATA-2 gene
expression in vivo, in situ hybridization analyses were performed. Embryos at early developmental stages were analyzed by whole-mount in
situ hybridization using both sense and antisense mGATA-2 probes. At
7.5 dpc, the expression of GATA-2 mRNA was first detected in the
lateral mesoderm, adjacent to the primitive streak, as well as in the
extraembryonic mesoderm (Fig 1A; panel B
shows a sense probe control). GATA-2 transcripts were also abundant in
the ectoplacental cone (data not shown) and para-aortic splanchnopleura
of 9.5 dpc embryos (Fig 1C; panel D shows a sense probe control). We
also observed the positive signals in the branchial arch and neural tissues. Evaluation of coronal sections of 9.5 and 10.5 dpc embryos (Fig 1E and F, respectively) allowed us to further localize the cellular distribution of GATA-2 mRNA in the splanchnopleura and the
mesoderm of the AGM region. GATA-2 expression was evident in rounded
cells bordering the dorsal aorta (Fig 1F). These results suggested that
mGATA-2 could be expressed in definitive hematopoietic precursors34,35,40 from the very earliest developmental
stages. However, we failed to detect GATA-2 mRNA in the yolk sac, which is the site of the developing primitive hematopoietic system (data not
shown).
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| Fig 1.
In situ hybridization analysis of GATA-2 mRNA expression
in early mouse embryos. GATA-2 mRNA expression was observed in the
lateral mesoderm adjacent to primitive streak of 7.5 dpc embryo (A and
B) and AGM region in 9.5 dpc embryo (C and D) with antisense probe (A
and C), but not with sense probe (B and D). Arrowhead in panel A
indicates the lateral mesoderm, and in panel C an arrow shows the
splanchnopleura. Panels E and F are coronal sections of 9.5 and 10.5 dpc embryos analyzed with the antisense probe. Abbreviations are a,
dorsal aorta; m, mesonephros; n, neural tube. Original magnifications
are ×50 (E) and ×25 (F).
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We next examined the expression of GATA-2 in the fetal liver and
postnatal spleen, which are two major definitive hematopoietic tissues,
by in situ hybridization. GATA-2 mRNA was easily detectable in 12.5 dpc
fetal livers in the section (Fig 2A).
Evaluation of GATA-2 expressing cells indicated that GATA-2 expressing
cells constituted 9.4% of total fetal liver cells at 12.5 dpc
(Table 1). The number of cells that
expressed GATA-2 declined postnatally (Fig 2B and C and Table 1),
concomitant with waning hematopoietic activity in the liver. GATA-2
signals were also observed in the red pulp of the neonatal mouse
spleen, another hematopoietic organ (Fig 2D and E). In the spleens of
5-day old pups, approximately 2% of the cells in the red pulp were
positive for GATA-2 expression. It is noteworthy that GATA-2 mRNA was
detected only in the red pulp, but not the white pulp, of the spleen
(Fig 2D). Because the white pulp consists principally of
lymphocytes,41 this observation is consistent with the
previous conclusion that GATA-2 is an important factor for myeloid
lineage differentiation, but perhaps less so for later lymphoid
differentiation.42 GATA-2-expressing cells were scarcely
detectable in the adult spleen (data not shown).
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| Fig 2.
Hematopoietic cell-specific expression of GATA-2 mRNA in
the liver and spleen of embryos and neonates. The expression of GATA-2
mRNA in the fetal liver of 12.5 dpc embryo (A) as well as in the liver
of 3-day (B) or 7-day (C) postnatal pups was analyzed by in situ
hybridization analysis on tissue sections. In 5-day old pups, GATA-2
mRNA-positive cells were observed in the red pulp but not in the white
pulp (R and W, respectively) of the spleen (D and E). The results with
antisense (A-E) and sense (F) probes are shown. Arrowheads in panels A,
B, D, and E indicate positive cells. Original magnifications are ×64
(A-C, E) and ×20 (D and F).
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From these in situ analyses, we also found that GATA-2 was expressed in
a variety of other embryonic and adult tissues. Amongst these sites,
GATA-2 mRNA was most abundantly expressed in neural tissues, especially
in the anterior horn of the developing spinal cord and dorsal root
ganglia in 17.5 dpc embryo
(Fig 3A), as well as in the
cerebral cortex (Fig 3B) and Purkinje cells in the cerebellum (Fig 3C)
of the adult mouse.
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| Fig 3.
GATA-2 mRNA expression in neuronal cells.
GATA-2 was expressed in anterior horn of the spinal cord and the dorsal
root ganglia of a 17.5 dpc embryo (A). In 12-week-old adult mice,
GATA-2 was expressed in the cerebral cortex (B) and Purkinje cells of
the cerebellum (arrowhead; C). Original magnifications are ×10 (A),
×64 (B), and ×16 (C).
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Three major DNase I hypersensitive sites surround the mGATA-2 IS
exon.
DNase I hypersensitive sites are generally considered to be strong
primary candidates for transcription regulatory
regions.1,37 To locate potential regulatory regions within
the mGATA-2 gene locus, we first performed DNase I
hypersensitivity assays using nuclei from P815 mouse mastocytoma cells,
which express abundant GATA-2. DNase I hypersensitive sites were
detected at 2.7 and 0.4 kbp 5' to the IS exon (shown as 3.1 and
0.8 kbp fragments in Fig 4A and B), as well
as one located 3.0 kbp 3' to the IS exon (shown as a 3.0 kbp
fragment in Fig 4C). Several weaker bands were also detected in these
experiments (Fig 4A and B).
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| Fig 4.
DNase I hypersensitive sites in the upstream region of
the IS and IG exons of GATA-2 gene. The top panel shows the
position of DNase I hypersensitive sites in the 5' region of the
locus. Long arrows represent major hypersensitive sites and short
arrows represent minor sites. Probe positions are also shown (a and b).
Genomic DNA treated with DNase I were digested by
KpnI/SacI (A),
XhoI/SacI (B), or
NotI/SalI (C), and hybridized with probe
a (A and B) or probe b (C). Two major hypersensitive sites were found
in the upstream region of the IS exon and two minor sites were also
found at 1.8 and 1.5 kbp upstream from the SacI site.
The SacI site is 0.4 kbp downstream from the
transcription start site of IS exon. One major hypersensitive site
exists in 3.0 kbp downstream from the NotI site in the
IS exon (C).
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GATA-2-directed LacZ reporter gene expression in hematopoietic
cells.
To test whether any of the DNase I hypersensitive sites identified in
the previous section have transcriptional regulatory activity, we
generated a series of reporter plasmids for transgenic analysis
(Fig 5). We first analyzed lines bearing a
transgene in which a 6.0 kbp genomic fragment 5' to the IS exon
was ligated to the LacZ gene (Fig 5).
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| Fig 5.
Reporter constructs used in the transgenic assays. Three
different LacZ reporter constructs, and one GFP construct, were
prepared to study the IS promoter mediated transcriptional activity of
the mGATA-2 gene. The open boxes represent the LacZ or
GFP gene and closed boxes represent mGATA-2 gene exons.
Abbreviations for the restriction enzyme sites are E, EcoRI; K,
KpnI; S, SalI; Xb, XbaI;
Xh, XhoI.
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Whole-mount staining of 9.5 dpc embryos from 6.0ISLacZ line 869 showed
that LacZ activity was detected in the para-aortic splanchnopleura
(Fig 6A; Fig 6B shows a nontransgenic
littermate). Two of five 6.0ISLacZ transgenic lines displayed similar
expression (Table 2). When this transgenic
embryo was sectioned saggitally, LacZ-positive cells were present in
the aortic wall (Fig 6C and D, arrows). Because only two out of five
6.0ISLacZ lines showed LacZ activity in the splanchnopleura, the
transcriptional activity of this region may be insufficient to overcome
the suppressive environment at the transgene integration sites (Table
2).
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| Fig 6.
Hematopoietic and neural tissue-specific transcription in
the 6.0ISLacZ transgenic mouse. Whole-mount LacZ staining of 9.5 (A and
B) and 11.5 (E and F) dpc embryos with 6.0ISLacZ transgene (line 869)
is shown. Panel B shows a transgene-negative littermate (9.5 dpc), and
C and D is a sagittal section of A. Panel D is a higher magnification
of C. Original magnifications are ×25 (C), and ×200 (D). Panel F is
a dorsal view of the embryo in panel E, showing that LacZ
reporter-positive cells reside in the anterior horn of the spinal cord.
Upper limb buds were eliminated to show the staining in the trunks.
Abbreviations: a, dorsal aorta; s, somites; n, neural tube.
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We also examined LacZ expression in 11.5 dpc embryos of 6.0ISLacZ line
869. On whole-mount staining, LacZ reporter activity was found in
neuronal cells and in the liver and heart of these embryos (Fig 6E and
F). Although the embryonic liver and heart appeared to be positively
stained in this embryo, signals could not be detected in the sections
(data not shown). We had previously observed similar low-level
transgene expression in the embryonic liver and heart.20
Because the regulatory elements controlling GATA-2
transcription in hematopoietic and neural tissues appeared to reside
within the 6.0ISLacZ transgene, we focused on further defining these elements by generating two smaller reporter gene constructs (Fig 5).
Examination of 9.5 and 11.0 dpc litters from six lines of 2.5ISLacZ and
two lines of 0.65ISLacZ transgenic mice indicated that all
hematopoietic tissues were negative for transgene expression (Table 2).
The 3.5 kbp differing between the two largest transgenes also contains
one of the strong DNase I hypersensitive sites (see Fig 4). Taken
together, these results indicate that a GATA-2 hematopoietic enhancer,
which is active in the splanchnopleura of 9.5 dpc embryos, resides
within the 3.5 kbp KpnI/XbaI fragment defining the
5' boundaries of the 6.0 and 2.5 kbp IS-LacZ transgenes.
The IS promoter contributes to GATA-2 expression in the para-aortic
splanchnopleura.
To further characterize the regulatory influence of this distal
promoter, GFP reporter transgenic mice were prepared that contained 7.0 kbp flanking the IS first exon (7.0ISGFP, see Fig 5). The use of the
GFP reporter enabled us to sensitively analyze live hematopoietic
cells. Two lines of 7.0ISGFP mice (398 and 982) displayed expression
that was similar to that observed in the 6.0ISLacZ transgenic lines in
both hematopoietic as well as neural tissues.
In 8.5 dpc 7.0ISGFP transgenic embryos, a ring of green fluorescence
was observed in the yolk sac (Fig 7A). We
previously observed a similar expression pattern when a LacZ reporter
gene was placed under the control of a GATA-1 transgene.10
Thus, the expression profiles of GATA-2 and GATA-1
genes appeared to overlap at this developmental stage. However,
although GATA-1-LacZ gene expression persists and expands after 8.5 dpc, the number of GATA-2-directed GFP-positive cells markedly
diminished in the yolk sac blood islands after this stage (data not
shown).
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| Fig 7.
Hematopoietic and neural tissue-specific transcription in
the 7.0ISGFP transgenic mouse. Whole-mount analysis of GFP expression
in 7.0ISGFP transgenic mouse embryos (line 398). GFP-positive cells
were identified in the 8.5 dpc yolk sac (A) and 9.0 (B), 9.5 (C), 10.5 (D), and 11.5 (E) dpc embryos. Green fluorescence-positive cells are
observed in para-aortic splanchnopleura (red arrows), liver rudiment
(white arrows), and vitelline vessels (arrowheads). In panel E , the
GFP brightly positive cells are in the ventral side of the neural tube.
Panel F shows a dorsal view of the embryo in panel E. These embryos
were also analyzed after dissection. GFP-positive cells are observed in
para-aortic splanchnopleura in 9.5 dpc embryo (G) and in or near the
dorsal aortic wall of 10.5 dpc embryo (H). GFP-positive cells are also
observed in 11.5 dpc fetal liver (I). Upper limb buds were eliminated
to show the staining in the trunks.
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Within the embryo proper, GFP staining was first visible in the
para-aortic splanchnopleura and liver rudiment at 9.0 (Fig 7B) and 9.5 (Fig 7C) dpc, which concurs with the 6.0ISLacZ transgenic analysis (see
Fig 6A). GFP expression was not detected earlier than 8.5 dpc in the
embryo (data not shown). Interestingly, we also detected GFP-positive
cells in the vitelline vessels (Fig 7B and C, arrowheads) of 9.0 and
9.5 dpc transgenic embryos. This observation suggests possible
circulation-mediated transfer of hematopoietic progenitors between the
yolk sac and the embryo proper. In the 10.5 dpc embryo, the liver
rudiment also contained GFP-positive cells (Fig 7D, arrow), but the
fluorescence was much weaker than that in neural tissues. In 11.5 dpc
embryos, GFP expression in neural tissues was again most prominent,
showing excellent coincidence with the 6.0ISLacZ analysis (Fig 7E and
F; compared with Fig 6D and E). In contrast, GFP-positive cells were
not as obvious in the AGM region nor in the fetal liver in the
whole-embryo analysis (Fig 7E).
The low frequency of the GFP-positive cells in hematopoietic tissues of
10.5 and 11.5 dpc transgenic embryos might reflect the limit of
whole-embryo analysis. We therefore dissected these transgenic embryos
to visualize cellular resolution of green fluorescence. We found
GFP-positive cells in 9.5 dpc para-aortic splanchnopleura (Fig 7G,
arrow), 10.5 dpc AGM region (Fig 7H), and 11.5 dpc fetal liver (Fig
7I). An important finding here was the identification of green
fluorescence-positive cells in or near the dorsal aortic wall (Fig 7H),
consistent with the in situ hybridization analysis (Fig 1F) and
analysis of the 6.0ISLacZ transgenic mouse (Fig 6C).
To determine frequency of these GFP-positive cells, the yolk sac, AGM,
and the fetal liver rudiment were dissected from the 10.5 dpc
transgenic embryos. Single-cell suspensions were then analyzed by FACS.
GFP-positive cells were found to constitute 0.5% of the yolk sac cells
(Fig 8A), 1% to 5% of
total liver cells, and 0.7% to 5% of total AGM cells at this stage
(data not shown). In contrast to the 10.5 dpc AGM, GFP-positive cells
represented only 0.25% of the total cells recovered from 13.5 dpc
transgenic embryo livers (data not shown), suggesting that the
expression of GFP reporter gene in this line is either subject to
position effect or that some activating cis-acting elements are
still missing in this transgene construct.
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| Fig 8.
GFP-positive cells show cell surface markers
characteristic to hematopoietic progenitors. The 7.0ISGFP transgenic
mouse embryos (line 398) were analyzed. Yolk sac from 10.5 dpc embryos
(A), AGM regions from 10.5 dpc embryos (B and C), and liver rudiments
from 10.5 and 11.0 dpc embryos (B and C) were dissected, and
single-cell suspensions from the tissues were analyzed by FACS directly
(A) or after staining with monoclonal antibodies (B and C).
PE-conjugated anti-Sca1, anti-CD45, and anti-c-kit antibodies (B), and
biotin-conjugated anti-CD34, anti-flk-1, and anti-Ter119 antibodies
and streptoavidine-APC were used (C). IgG2b antibody was used as a
control.
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We next stained 10.5 dpc AGM and liver cells (line 398) with antibodies
recognizing cell surface markers, and performed FACS analysis. A
fraction of the GFP-positive cells in the liver and AGM were stained
with the anti-CD45 (common leukocyte antigen) or anti-c-kit antibodies
(Fig 8B, compare upper right boxes of TG(+) with TG(-)). GFP/CD45 and
GFP/c-kit double-positive cells are more abundant in 10.5 and 11.0 dpc
fetal liver than in the 10.5 dpc AGM region. The 10.5 dpc AGM contained
GFP/CD34 double-positive cells, whereas the 10.5 dpc liver cells
contained GFP/CD34 and GFP/flk-1 double-positive cells (Fig 8C). We
were unable to detect GFP/Ter119 (mature erythroid marker)
double-positive cells (Fig 8C). In the 11.0 dpc fetal liver,
approximately 40% of the GFP-positive cells are also immunoreactive
with anti-CD45 antibody (Fig 8B). These data thus indicate
hematopoietic progenitors are clearly among the GATA-2-directed GFP
positive cells.
We then sorted the GFP-positive and -negative fetal liver cells from
11.5 dpc transgenic embryos and extracted RNA from both cell fractions.
To assess endogenous GATA-2 gene expression, a promoter-specific RT-PCR analysis was performed. The assay detected PCR
products containing the IS exon sequence in the GFP-positive fraction,
but not in the GFP-negative fraction (Fig
9), indicating that the 7.0 kbp gene regulatory region has an activity
to recapitulate the IS promoter activity in 11.5 dpc hematopoietic
progenitors.
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| Fig 9.
GFP-expression recapitulates endogenous IS promoter
activity. GFP-positive and -negative cell fractions were sorted by FACS
from the liver of 11.5 dpc embryo of 7.0ISGFP transgenic mouse.
Endogenous expression of the IS exon of mouse GATA-2 gene was analyzed
using RT-PCR. The primer pairs amplifying the elongation factor (EF)
1 mRNA was used as a control.38
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The IS promoter contributes to neural expression of GATA-2.
The IS promoter was also used to direct GATA-2 neural expression. LacZ
activity was observed in the adult brain of 6.0ISLacZ, 2.5ISLacZ, and
0.65ISLacZ transgenic mice, the expression profiles of these transgenes
were generally consistent with that determined by the in situ
hybridization analysis (Table 2, Fig 3). The neural expression of GFP
was also detected in the 7.0ISGFP transgenic embryos (see Fig 7D to F).
Dissection of transgenic 11.5 dpc embryos showed that GFP was expressed
in the ventral portion of neural tube, consistent with the results of
the in situ hybridization analysis (Fig 3A). In a higher magnification
of a 9.5 dpc embryo, large filamentous GFP-positive cells are visible
in the dorsal region (Fig 7G). These results suggest that a positive
neural regulatory element is located within the 0.65 kbp region, lying immediately 5' to the IS exon, in contrast to the regulation of GATA-2 gene expression in hematopoietic cells (see above).
These data thus show that the elements regulating the usage of
GATA-2 gene IS promoter activity in hematopoietic as well as
neural cells are under the regulation of multiple regulatory elements.
| |
DISCUSSION |
Transgenic mouse analysis is an effective tool for defining the
position and identity of cis-acting elements that function in
restricted tissue types or at different stages of
development.10 To identify hematopoietic regulatory
elements for the GATA-2 gene, we examined the activity of
various segments of the mGATA-2 gene. In this in vivo analysis,
we never detected ectopic expression of the reporter genes, so tissues
in which these activities were observed are likely to be sites in which
the regulatory regions in the transgene constructs exert their normal
activity. Concurrent in situ analyses underscored this contention.
The cis-regulatory elements that direct GATA-2 expression in
the AGM region and neural tissues were identified here, although these
genomic sequences alone appeared to be insufficient to fully
recapitulate the endogenous expression profile of the GATA-2 gene.
In this study, we identified GATA-2-expressing cells in the
para-aortic splanchnopleura in 9.5 dpc and AGM region in 10.5 dpc mouse
embryos. It is significant to note that the definitive hematopoietic
lineage was shown to arise from mesodermal cells in these regions of
the embryo.34,35 These hematopoietic cells of 10.5 dpc AGM
region retain both spleen colony forming activity and long-term
reconstitution ability in lethally irradiated mice.34 In
this regard, GATA-2 and SCL/tal-1 mRNAs were reportedly expressed in
lineage marker-negative (Lin ) cells isolated from
7.0 dpc early- to mid-primitive streak stage embryos when cocultured
with stromal cells for 5 to 6 days, and this fraction contains the
earliest detectable multipotent progenitors.43 GATA-2-positive cell lines derived from this culture system display long-term reconstituting activity of the lymphohematopoietic system in
lethally irradiated mice.43 Hematopoietic stem cells in the AGM region and liver of mouse embryos have been found in the
c-kit+/CD34+ double-positive
fraction.44 Cells expressing flk-1 in 10.5-11.5 dpc AGM
region have been shown to generate both hematopoietic and angiopoietic
cells, so that these cells are termed hemangioblasts, or hematogenic
endothelial cells.45,46 The present study of 7.0ISGFP
transgenic mouse embryos showed that the GATA-2-GFP-positive hematopoietic cell fraction contains CD34, c-kit, and flk-1-positive cells. Additionally, we previously showed that GATA-2 mRNA is expressed
in the Lin/c-kit+/Sca1+
fraction of adult bone marrow cells, which contains the highest percentage of long-term reconstituting hematopoietic stem
cells.14,31 Taken together, these observations suggest that
GATA-2 activity is a common feature of hematopoietic stem cells and
progenitors.22-24,31
We found in this analysis that GATA-2 mRNA is first detected in the
extra-embryonic and lateral plate mesoderm, and later in the
para-aortic splanchnopleura, the AGM region, and the liver rudiment,
during mouse development. GFP expression was also detected in the early
yolk sac cells, and diminished in later stages. These observations are
consistent with a previous report concluding that GATA-2 mRNA is
expressed in the late primitive streak stage mesoderm, and is then
downregulated in yolk sac mesodermal cells.40 On the other
hand, both the expression of GATA-1 and GATA-1 gene regulatory
region-directed LacZ reporter genes are detected abundantly in the
visceral yolk sac blood islands.10 The 9.5 to 10.5 dpc yolk
sac hematopoietic cells of GATA-2 knockout mice show impaired hematopoietic ability, but 15% to 50% of the normal number of primitive erythrocytes are recovered from GATA-2 ( /)
embryos.12 These observations suggest that GATA-2 is
required only at the beginning of hematopoietic cell differentiation in
yolk sac hematopoiesis, and that a requirement for GATA-2 at this stage
may be compensated to some extent, or even circumvented, by other factors.
Previous transient transfection assays indicated that both IS and IG
promoter activities are regulated by regions lying within 100 bp from
their respective transcription start sites, and these assays failed to
identify any additional transcriptional regulatory requirements in
other regions of the gene.31 In contrast, transgenic mice
bearing reporters that contain additional upstream sequence showed
reporter gene expression in hematopoietic tissues. The most
straightforward way to explain this discrepancy is to invoke chromatin
or nucleosome remodeling activity,47-49 which might be detectable only in chromatin-associated (ie, integrated) transgenes. Indeed, specific cis-acting elements have been defined with the ability to ensure an open chromatin configuration.50, 51 An
alternative explanation for this disparity is that hematopoietic stem
cells, or early progenitors, are by nature very difficult to mimic in
transformed cell culture assays, and therefore enhancer activities that
function only in a very specific subpopulation of early hematopoietic
cells might be difficult to reproduce in cell culture.
The transcriptional activity of the 6.0 and 7.0 kbp sequences upstream
of the IS promoter was detected in the ventral central nervous system.
In this regard, it is interesting to note that GATA-2 is expressed
during pituitary organogenesis.52, 53 The expression of
GATA-2 in the embryonic pituitary gland is reported to be under the
control of BMP-2/4 signals,53 which are also regulators of
mesodermal development.54,55 BMP-4 has been shown to
stimulate the expression of GATA-2 and induce hematopoietic development
in Xenopus.30 Therefore, the possibility exists that the transcription of IS-initiated GATA-2 transcription may be
under the influence of BMP-2/4 signaling.
In summary, we have shown here that the expression of GATA-2 in
hematopoietic cells and neural tissues is regulated by discrete genomic
regions that direct GATA-2 gene IS promoter-directed expression in
transgenic mice. The identification of a regulatory region that can
direct para-aortic splanchnopleura and AGM expression of the
GATA-2 gene may enable targeted expression of various factors to hematopoietic stem cells in vivo. Furthermore, this information may
enable us to isolate specific hematopoietic progenitor cells, perhaps
including the most primitive stem cells, using the GATA-2 gene
regulatory region, and we are currently exploring this exciting prospect.
| |
ACKNOWLEDGMENT |
We thank Drs Kim-Chew Lim, Ken-ichi Yagami, Naomi Kaneko, Hozumi
Motohashi, Takahiko Hara, Yousuke Mukouyama, and Hiromitsu Nakauchi for
critical reading of the manuscript and experimental assistance. We also
thank Drs Kazuhiko Umezono, Shin-ichi Nishikawa, and Atushi Ichikawa
for generous gifts of pCMX-SAH/Y145F plasmid, anti-flk-1 antibody and
P815 cell line, respectively.
| |
FOOTNOTES |
Submitted February 24, 1998; accepted February 17, 1999.
N.M. and J.O. contributed equally to this work
Supported in part by a research grant from the NIH (GM28896; J.D.E.);
Grants-in-Aid from the Ministry of Education, Science, Sports, and
Culture; CREST; and the Japanese Society for Promotion of Sciences
(JSPS Research for the Future Project).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Masayuki Yamamoto, Institute of Basic Medical
Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8575, Japan; e-mail: masi{at}tara.tsukuba.ac.jp.
| |
REFERENCES |
1.
Engel JD, George KM, Ko LJ, Kornhauser JM, Leonard MW, Ting P, Yamamoto M:
Transcription factor regulation of hematopoietic cells.
Semin Hematol
28:158, 1991[Medline]
[Order article via Infotrieve]
2.
Yamamoto M, Takahashi S, Onodera K, Muraosa Y, Engel JD:
Upstream and downstream of erythroid transcription factor GATA-1.
Genes Cells
2:107, 1997[Abstract]
3.
Evans T, Felsenfeld G:
The erythroid-specific transcription factor Eryf1: A new finger protein.
Cell
58:95, 1989[Medline]
[Order article via Infotrieve]
4.
Romeo PH, Prandini MH, Joulin V, Mignotte V, Prenant M, Vainchenker W, Marquerie G, Uzan G:
Megakaryocytic and erythrocytic lineages share specific transcription factor.
Nature
344:447, 1990[Medline]
[Order article via Infotrieve]
5.
Tsai SF, Martin DIK, Zon LI, D'Andrea AD, Wong GG, Orkin SH:
Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells.
Nature
339:446, 1989[Medline]
[Order article via Infotrieve]
6.
Takahashi S, Onodera K, Motohashi H, Suwabe N, Hayashi N, Yanai N, Nabesima Y, Yamamoto M:
Arrest in primitive erythroid cell development caused by promoter-specific disruption of the GATA-1 gene.
J Biol Chem
272:12611, 1997[Abstract/Free Full Text]
7.
Fujiwara Y, Browne CP, Cunniff K, Goff SC, Orkin SH:
Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1.
Proc Natl Acad Sci USA
93:12355, 1996[Abstract/Free Full Text]
8.
Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin SH:
A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development.
EMBO J
16:3965, 1997[Medline]
[Order article via Infotrieve]
9.
Ito E, Toki T, Ishihara H, Ohtani H, Gu L, Yokoyama M, Engel JD, Yamamoto M:
Erythroid transcription factor GATA-1 is abundantly transcribed in mouse testis.
Nature
362:466, 1993[Medline]
[Order article via Infotrieve]
10.
Onodera K, Takahashi S, Nishimura S, Ohta J, Motohashi H, Yomogida K, Hayashi N, Engel JD, Yamamoto M:
GATA-1 transcription is controlled by distinct regulatory mechanisms during primitive and definitive erythropoiesis.
Proc Natl Acad Sci USA
94:4487, 1997[Abstract/Free Full Text]
11.
Onodera K, Yomogida K, Suwabe N, Takahashi S, Muraosa Y, Hayashi N, Ito E, Gu L, Rassoulzadegan M, Engel J, Yamamoto M:
Conserved structure, regulatory elements, and transcriptional regulation from the GATA-1 gene testis promoter.
J Biochem
121:251, 1997[Abstract/Free Full Text]
12.
Tsai FY, Keller G, Kuo FC, Weiss M, Chen J, Rosenblatt M, Alt FW, Orkin SH:
An early haematopoietic defect in mice lacking the transcription factor GATA-2.
Nature
371:221, 1994[Medline]
[Order article via Infotrieve]
13.
Yamamoto M, Ko LJ, Leonard MW, Beug H, Orkin SH, Engel JD:
Activity and tissue-specific expression of the transcription factor NF-E1 [GATA] multigene family.
Genes Dev
4:1650, 1990[Abstract/Free Full Text]
14.
Nagai T, Harigae H, Ishihara H, Motohashi H, Minegishi N, Tsuchiya S, Hayashi N, Gu L, Andres B, Engel JD, Yamamoto M:
Transcription factor GATA-2 is expressed in erythroid, early myeloid, and CD34+ human leukemia-derived cell lines.
Blood
84:1074, 1994[Abstract/Free Full Text]
15.
Walmsley ME, Guille MJ, Bertwistle D, Smith JC, Pizzey JA, Patient RK:
Negative control of Xenopus GATA-2 by activin and noggin with eventual expression in precursors of the ventral blood islands.
Development
120:2519, 1994[Abstract/Free Full Text]
16.
Zon LI, Mather C, Burgess S, Bolce ME, Harland RM, Orkin SH:
Expression of GATA-binding proteins during embryonic development in Xenopus laevis.
Proc Natl Acad Sci USA
88:10642, 1991[Abstract/Free Full Text]
17.
Detrich HW III, Kieran MW, Chan FY, Barone LM, Yee K, Rundstadler JA, Pratt S, Ransom D, Zon LI:
Intraembryonic hematopoietic cell migration during vertebrate development.
Proc Natl Acad Sci USA
92:10713, 1995[Abstract/Free Full Text]
18.
Lee ME, Temizer DH, Clifford JA, Quertermous T:
Cloning of the GATA-binding protein that regulates endothelin-1 gene expression in endothelial cells.
J Biol Chem
266:16188, 1991[Abstract/Free Full Text]
19.
Meng A, Tang H, Ong BA, Farrell MJ, Lin S:
Promoter analysis in living zebrafish embryos identifies a cis-acting motif required for neuronal expression of GATA-2.
Proc Natl Acad Sci USA
94:6267, 1997[Abstract/Free Full Text]
20.
Zhou Y, Lim KC, Onodera K, Takahashi S, Ohta J, Minegishi N, Tsai FY, Orkin SH, Yamamoto M, Engel JD:
Rescue of the embryonic lethal hematopoietic defect reveals a critical role for GATA-2 in urogenital development.
EMBO J
17:6689, 1998[Medline]
[Order article via Infotrieve]
21.
Mouthon MA, Bernard O, Mitjavila MT, Romeo PH, Vainchenker W, Mathieu MD:
Expression of tal-1 and GATA-binding proteins during human hematopoiesis.
Blood
81:647, 1993[Abstract/Free Full Text]
22.
Orlic D, Anderson S, Biesecker LG, Sorrentino BP, Bodine DM:
Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukin 5 and 7.
Proc Natl Acad Sci USA
92:4601, 1995[Abstract/Free Full Text]
23.
Labbaye C, Valtieri M, Barberi T, Meccia E, Masella B, Pelosi E, Condorelli GL, Testa U, Peschle C:
Differential expression and functional role of GATA-2, NF-E2, and GATA-1 in normal adult hematopoiesis.
J Clin Invest
95:2346, 1995
24.
Leonard M, Brice M, Engel JD, Papayannopoulou T:
Dynamics of GATA transcription factor expression during erythroid differentiation.
Blood
82:1071, 1993[Abstract/Free Full Text]
25.
Leonard MW, Lim K-C, Engel JD:
Expression of the chicken GATA factor family during early erythroid development and differentiation.
Development
119:519, 1993[Abstract]
26.
Jippo T, Mizuno H, Xu Z, Nomura S, Yamamoto M, Kitamura Y:
Abundant expression of transcription factor GATA-2 in proliferating but not in differentiated mast cells in tissues of mice: Demonstration by in situ hybridization.
Blood
87:993, 1996[Abstract/Free Full Text]
27.
Zon L, Yamaguchi Y, Yee K, Albee EA, Kimura A, Bennett JC, Orkin SH, Ackerman SJ:
Expression of mRNA for the GATA-binding proteins in human eosinophils and basophils: Potential role in gene transcription.
Blood
81:3234, 1993[Abstract/Free Full Text]
28.
Partington GA, Bertwistle D, Nicolas RH, Kee WJ, Pizzey JA, Patient RK:
GATA-2 is a maternal transcription factor present in Xenopus oocytes as a nuclear complex which is maintained throughout early development.
Dev Biol
181:144, 1997[Medline]
[Order article via Infotrieve]
29.
Turpen JB, Kelley CM, Mead PE, Zon LI:
Bipotential primitive-definitive hematopoietic progenitors in the vertebrate embryo.
Immunity
7:325, 1997[Medline]
[Order article via Infotrieve]
30.
Maeno M, Mead PE, Kelley C, Xu R, Kung H, Suzuki A, Ueno N, Zon LI:
The role of BMP-4 and GATA-2 in the induction and differentiation of hematopoietic mesoderm in Xenopus laevis.
Blood
88:1965, 1996[Abstract/Free Full Text]
31.
Minegishi N, Ohta J, Suwabe N, Nakauchi H, Ishihara H, Hayashi N, Yamamoto M:
Alternative promoters regulate transcription of the mouse GATA-2 gene.
J Biol Chem
273:3625, 1998[Abstract/Free Full Text]
32.
Brewer AC, Guille MJ, Fear DJ, Partington GA, Patient RK:
Nuclear translocation of a maternal CCAAT factor at the start of gastrulation activates Xenopus GATA-2 transcription.
EMBO J
14:757, 1995[Medline]
[Order article via Infotrieve]
33.
Fleenor DE, Langdon SD, deCastro CM, Kaufman RE:
Comparison of human and Xenopus GATA-2 promoters.
Gene
179:219, 1996[Medline]
[Order article via Infotrieve]
34.
Medvinsky A, Dzierzak E:
Definitive hematopoiesis is autonomously initiated by the AGM region.
Cell
86:897, 1996[Medline]
[Order article via Infotrieve]
35.
Cumano A, Dieterlen-Lievre F, Godin I:
Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura.
Cell
86:907, 1996[Medline]
[Order article via Infotrieve]
36.
Harigae H, Takahashi S, Suwabe N, Ohtsu H, Gu L, Yang Z, Tsai F-Y, Kitamura Y, Engel JD, Yamamoto M:
Differential roles of GATA-1 and GATA-2 in growth and differentiation of mast cells.
Genes Cells
3:39, 1998[Abstract]
37.
Stalder J, Larsen A, Groudine M, Dolan M, Engel JD, Weintraub H:
Tissue-specific DNA cleavages in the globin chromatin domain introduced by DNase I.
Cell
20:451, 1980[Medline]
[Order article via Infotrieve]
38.
Matsuzaki Y, Nakayama K, Nakayama K, Tomita T, Isoda M, Loh DY, Nakauchi H:
Role of bcl-2 in the development of lymphoid cells from the hematopoietic stem cell.
Blood
89:853, 1997[Abstract/Free Full Text]
39.
Kataoka H, Takakura N, Nishikawa S, Tsichida K, Kodama H, Kunisada T, Risau W, Kita T, Nishikawa S-I:
Expression of PDGF receptor alpha, c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells.
Dev Growth Differ
39:729, 1997[Medline]
[Order article via Infotrieve]
40.
Silver LJ:
Initiation of murine embryonic erythropoiesis: A spatial analysis.
Blood
89:1154, 1997[Abstract/Free Full Text]
41.
Liu Y-J, de Bouteiller O, Fugier-Vivier I:
Mechanisms of selection and differentiation in germinal centers.
Curr Opin Immunol
9:256, 1997[Medline]
[Order article via Infotrieve]
42.
Minegishi N, Morita S, Minegishi M, Tsuchiya S, Konno T, Hayashi N, Yamamoto M:
Expression of GATA transcription factors in myelogenous and lymphoblastic leukemia cells.
Int J Hematol
65:239, 1997[Medline]
[Order article via Infotrieve]
43.
Palacios R, Imhof BA:
Primitive lymphohematopoietic precursor cell lines generated in culture from day 7 early-mid-primitive streak stage mouse embryo.
EMBO J
15:6869, 1996[Medline]
[Order article via Infotrieve]
44.
Sanchez MJ, Holmes A, Miles C, Dzierzak E:
Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo.
Immunity
5:513, 1996[Medline]
[Order article via Infotrieve]
45.
Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML, Schuh AC:
Failure of blood-island formation and vasculogenesis in FLK-1-deficient mice.
Nature
376:62, 1995[Medline]
[Order article via Infotrieve]
46.
Nishikawa S-I, Nishikawa S, Yoshida H, Kizumoto M, Kataoka H, Katsura Y:
In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos.
Immunity
8:761, 1998[Medline]
[Order article via Infotrieve]
47.
Gong QH, McDowell JC, Dean A:
Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the  -globin gene in vivo by 5' hypersensitive site 2 of the -globin locus control region.
Mol Cell Biol
16:6055, 1996[Abstract]
48.
Linhoff MW, Wright KL, Ting JP-Y:
CCAAT-binding factor NF-Y and RFX are required for in vivo assembly of a nucleoprotein complex that spans 250 base pairs: The invariant chain promoter as a model.
Mol Cell Biol
17:4589, 1997[Abstract]
49.
Tsukiyama T, Daniel C, Tamkun J, Wu C:
ISWI, a member of the SWI2/SNF2 ATPase family, encodes the 140 kDa subunit of the nucleosome remodeling factor.
Cell
83:1021, 1995[Medline]
[Order article via Infotrieve]
50.
Festebstein R, Tolaini M, Corbella P, Mamalaki C, Parrington J, Fox M, Miliou A, Jones M, Kioussis D:
Locus control region function and heterochromatin-induced position effect variegation.
Science
271:1123, 1996[Abstract]
51.
Walters MC, Fiering S, Eidemiller J, Magis W, Groudine M, Martin DIK:
Enhancers increase the probability but not the level of gene expression.
Proc Natl Acad Sci USA
92:7125, 1995[Abstract/Free Full Text]
52.
Gordon DF, Lewis SR, Haugen BR, James RA, McDermott MT, Wood WM, Ridgway EC:
Pit-1 and GATA-2 interact and functionally cooperate to activate the thyrotropin -subunit promoter.
J Biol Chem
272:24339, 1997[Abstract/Free Full Text]
53.
Treiner M, Gleiberman AS, O'Connell SM, Szeto DP, McMahon JA, McMahon AP, Rosenfeld MG:
Multistep signaling requirements for pituitary organogenesis in vivo.
Genes Dev
12:1691, 1998[Abstract/Free Full Text]
54.
Hogan BLM:
Bone morphogenic proteins: Multifunctional regulators of vertebrate development.
Genes Dev
10:1580, 1996[Free Full Text]
55.
Tonegawa A, Funayama N, Ueno N, Takahashi Y:
Mesodermal subdivision along the mediolateral axis in chicken controlled by different concentration of BMP-4.
Development
124:1975, 1997[Abstract]
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3954 - 3962.
[Abstract]
[Full Text]
[PDF]
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N. Minegishi, N. Suzuki, T. Yokomizo, X. Pan, T. Fujimoto, S. Takahashi, T. Hara, A. Miyajima, S.-i. Nishikawa, and M. Yamamoto
Expression and domain-specific function of GATA-2 during differentiation of the hematopoietic precursor cells in midgestation mouse embryos
Blood,
August 1, 2003;
102(3):
896 - 905.
[Abstract]
[Full Text]
[PDF]
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J. A. Grass, M. E. Boyer, S. Pal, J. Wu, M. J. Weiss, and E. H. Bresnick
GATA-1-dependent transcriptional repression of GATA-2 via disruption of positive autoregulation and domain-wide chromatin remodeling
PNAS,
July 22, 2003;
100(15):
8811 - 8816.
[Abstract]
[Full Text]
[PDF]
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P. Oettgen
Transcriptional Regulation of Vascular Development
Circ. Res.,
August 31, 2001;
89(5):
380 - 388.
[Abstract]
[Full Text]
[PDF]
|
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|
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C. J. Marshall, C. Kinnon, and A. J. Thrasher
Polarized expression of bone morphogenetic protein-4 in the human aorta-gonad-mesonephros region
Blood,
August 15, 2000;
96(4):
1591 - 1593.
[Abstract]
[Full Text]
[PDF]
|
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S. Nishimura, S. Takahashi, T. Kuroha, N. Suwabe, T. Nagasawa, C. Trainor, and M. Yamamoto
A GATA Box in the GATA-1 Gene Hematopoietic Enhancer Is a Critical Element in the Network of GATA Factors and Sites That Regulate This Gene
Mol. Cell. Biol.,
January 15, 2000;
20(2):
713 - 723.
[Abstract]
[Full Text]
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TOP
ABSTRACT
INTRODUCTION