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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 434-442
Role of GATA-1 in Proliferation and Differentiation of Definitive
Erythroid and Megakaryocytic Cells In Vivo
By
Satoru Takahashi,
Takuya Komeno,
Naruyoshi Suwabe,
Keigyo Yoh,
Osamu Nakajima,
Sigeko Nishimura,
Takashi Kuroha,
Toshiro Nagasawa, and
Masayuki Yamamoto
From the Institute of Basic Medical Sciences and Center for TARA and
the Division of Hematology, Institute of Clinical Medicine, University
of Tsukuba, Tsukuba, Japan
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ABSTRACT |
To elucidate the contributions of GATA-1 to definitive hematopoiesis
in vivo, we have examined adult mice that were rendered genetically
defective in GATA-1 synthesis (Takahashi et al, J Biol Chem
272:12611, 1997). Because the GATA-1 gene is located on the X
chromosome, which is randomly inactivated in every cell, heterozygous
females can bear either an active wild-type or mutant (referred to as
GATA-1.05) GATA-1 allele, consequently leading to variable
anemic severity. These heterozygous mutant mice usually developed
normally, but they began to die after 5 months. These affected animals
displayed marked splenomegaly, anemia, and thrombocytopenia. Proerythroblasts and megakaryocytes massively accumulated in the spleens of the heterozygotes, and we showed that the neomycin resistance gene (which is the positive selection marker in ES cells)
was expressed profusely in the abnormally abundant cells generated in
the GATA-1.05 mutant females. We also observed hematopoiesis outside of the bone marrow in the affected mutant mice. These data
suggest that a small number of GATA-1.05 mutant hematopoietic progenitor cells begin to proliferate vigorously during early adulthood, but because the cells are unable to terminally
differentiate, this leads to progenitor proliferation in the spleen and
consequently death. Thus, GATA-1 plays important in vivo roles for
directing definitive hematopoietic progenitors to differentiate along
both the erythroid and megakaryocytic pathways. The GATA-1 heterozygous mutant mouse shows a phenotype that is analogous to human
myelodysplastic syndrome and thus may serve as a useful model for this
disorder.
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INTRODUCTION |
TRANSCRIPTION FACTOR GATA-1 is expressed
within the hematopoietic hierarchy in erythroid, megakaryocytic,
eosinophilic, and mast cell lineages1-5 as well as in
Sertoli cells of the testis.6 The GATA-1 gene has been
mapped to the X chromosome7 and two alternative
promoters/first exons with differential tissue-specific usage have been
identified. The 5 IT promoter and the downstream IE promoter
dictate GATA-1 transcription in Sertoli cells and erythroid cells,
respectively.8
The functional roles of GATA-1 in hematopoietic cell development have
been analyzed by gene targeting (reviewed in Yamamoto et
al9). Analysis of chimeric animals generated by
GATA-1-null embryonic stem (ES) cells indicated that the mutant cells
do not contribute to the mature erythroid population.10 In
vitro, GATA-1-null ES cells are also unable to differentiate into
mature erythroid cells,11-13 thus suggesting that GATA-1 is
required for the terminal differentiation of committed erythroid
progenitor cells.
Because previous attempts at generating germ line GATA-1-null alleles
were unsuccessful,10 we prepared an erythroid
promoter-specific knock-down of the GATA-1 gene.14 Because
male embryos bearing this mutation expressed GATA-1 mRNAs at
approximately 5% of the level that accumulated in wild-type embryos,
we therefore referred to this mutant allele as GATA-1.05. All
mutant male embryos (of GATA-1.05/Y genotype) died by 12.5 days
post coitus (dpc) due to impaired primitive hematopoiesis. Analysis of
the male embryos (by specifically marking primitive progenitors with a
lacZ transgene) demonstrated that the maturation of these
progenitors was blocked at the proerythroblast stage in the
GATA-1.05/Y embryos, indicating that GATA-1 is necessary for
terminal differentiation during primitive erythropoiesis.14
This conclusion is consistent with that of another recent analysis
studying GATA-1-null mutant embryonic erythropoiesis.15
However, the in vivo functional contributions of GATA-1 to definitive
hematopoiesis are still unknown, because mutant male mice
(GATA-1.05/Y) die before full commencement of definitive hematopoiesis due to a severe deficit in primitive hematopoiesis. To
address this question, we took advantage of the fact that GATA-1 gene
is X-linked,7 and because of random inactivation of the X
chromosome (ie, Lyonization16) in somatic tissues,
heterozygous mutant females should always be chimeric for GATA-1 gene
expression. Analysis of heterozygous (GATA-1.05/X) female mice
showed that GATA-1 is required for the terminal differentiation of
definitive erythroid and megakaryocytic cells. These results indicate
that GATA-1 is a key regulator of definitive hematopoietic cell
differentiation in vivo and suggest that the loss of GATA-1 is an
accelerating factor for abnormal expansion of erythroid and
megakaryocytic lineage progenitor cells.
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MATERIALS AND METHODS |
Mice.
Mice bearing the GATA-1.05 allele15 were bred in
clean rooms at the Animal Research Institute of Tohoku University and
in the Animal Research Center at the University of Tsukuba (Tsukuba, Japan). Hematological examination of mice heterozygous for the GATA-1.05 allele was performed by phlebotomy from the
retro-orbital plexus. After initially noting that adult heterozygous
mutant female mice have abnormally shorter life spans, they were
monitored carefully after becoming anemic so that necropsies could be
performed within 12 hours after death. Biopsy of overtly healthy
GATA-1.05 heterozygous mutant females was performed on a group
of 11 young mice of 8 to 12 weeks of age.
Histological analysis of mice.
Samples of major organs of the GATA-1.05 heterozygous mice and
their littermates were obtained either by necropsy or biopsy. The
samples were fixed in 10% buffered formalin and embedded in paraffin.
Sections were stained with hematoxylin and eosin for histological
examination. Cryo-sections were processed for immunohistochemistry with
N6 anti-GATA-1 antibody6 and antimouse glycoprotein IIb antibody (Komeno et al, submitted for publication).
Acetylcholinesterase staining for megakaryocytes was performed as
described.17
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of
GATA-1 and neomycin resistant gene expression.
RNA was isolated from spleens using the guanidine thiocyanate/cesium
chloride ultracentrifugation method.18 First-strand cDNA
was synthesized using Superscript reverse-transcriptase (GIBCO BRL,
Gaithersburg, MD) at 37°C for 1 hour, and 1 µL of
this 20 µL reaction mixture was used for the PCR reactions. Amplified products were analyzed on 2% agarose gels. Sequences of the primers used were as follows: erythroid-specific GATA-1 transcript (expected size, 483 bp), 5-TAAGGTGGCTGAATCCTCTGCATC and
3-CCGTCTTCAAGGTGTCCAAGAACGT; testis-specific GATA-1
transcript (expected size, 529 bp), 5-CGTGAAGCGAGACCATCGTC and
the 3-primer was the same as the erythroid 3-primer; and neomycin phosphotransferase (expected product, 339 bp),
5-AAGTATCCATCATGGCTGATG and 3-TAGCCAACGCTATGTCCTGATA.
Primers for glucose-6-phosphate dehydrogenase (G6PD; expected size, 162 bp) were synthesized as described previously.19
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RESULTS |
Female mice of GATA-1.05/X genotype have a shortened life span.
We have previously reported that GATA-1.05/X mutant female mice
suffered varying degrees of anemia in the embryonic and early neonatal
stages.14 Because the wild-type siblings displayed none of
these symptoms, we suspected that this heterogeneity in phenotype was
probably due to variable extent of random inactivation of the wild-type
X chromosome.16 We therefore selected 19 heterozygous and 8 wild-type female mice and followed their life span. The GATA-1.05/X mice had a shortened life span in comparison to
wild-type littermates. More than half of the heterozygous mutant female mice died within 7 months after birth (Fig
1), whereas all 8 control female animals survived beyond the test
period. Thus, the GATA-1.05 lesion leads to a dramatically
foreshortened life span in mature heterozygous females.
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| Fig 1.
Cumulative mortality in GATA-1.05 heterozygous
( ) and wild-type ( ) female mice. Heterozygous GATA-1.05
females14 were mated with wild-type and the resultant
female progenies were first genotyped. The life span of 19 heterozygous
mutant and 8 wild-type litter mates was then examined.
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Anemia and thrombocytopenia in adult GATA-1.05 female mutant
mice.
To elucidate the cause of death in the heterozygous mutant mice, we
dissected and analyzed the moribund animals. We found that most of them
displayed marked splenomegaly (varying principally in degree) and that
the appearance of the enlarged spleen was light red to pink in color,
rather than the typical dark red color (Fig 2A).
Furthermore, expansion of the spleen was so pronounced that other
abdominal organs were compressed, as is also often seen in human
myeloproliferative disorders.
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| Fig 2.
Splenomegaly in the GATA-1.05 heterozygous mutant
female mice. (A) Spleens were removed from wild-type (upper) and
GATA-1.05 heterozygous (lower) littermates after genotyping and
hematocrit screening of animals. Note that the spleen of the
GATA-1.05 heterozygous mouse is markedly enlarged and is
somewhat pink as compared with the dark red spleen from the wild-type
mouse. (B) Sizes of the spleens from 6 wild-type (wt) and 22 heterozygous mutant (+/GATA-1.05) female mice. +/ b
shows the spleen weights of the biopsied 8- to 12-week-old healthy
GATA-1.05 heterozygous mice (those listed in Table 1). +/
a indicates mice that were found dead whose spleen weights were
determined upon necropsy. ( ) Mice no. 12 through 14 of Table 1 are
indicated.
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| Fig 4.
Acetylcholinesterase and anti-glycoprotein IIb staining
of megakaryocytes in the spleens of female mice. Megakaryocytes in a
heterozygous mutant spleen (C and D) were positive for
acetylcholinesterase staining. The appearance of the positive staining
cells is morphologically similar to megakaryocytes in a wild-type
spleen (A and B). Note that there are more megakaryocytes in the mutant
mouse spleen. These cells were also positive for glycoprotein IIb (E
and F). Original magnifications × 20 (A, C, and E) and × 80 (B, D,
and F).
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Eight litters from GATA-1.05 mutant female and wild-type male
mice crosses either by biopsy or autopsy soon after death were examined. We found that all of the mice that died spontaneously were of
the GATA-1.05/X genotype and that most of these showed enlargement of the spleen to variable degrees (Fig 2B, +/ a). A
second group of 11 8- to 12-week-old female mutant mice were killed,
and their spleens were examined by biopsy. These mice were considerably
younger than the previous set of mutant females examined that had died
spontaneously. We again observed slight enlargement of the spleen among
these overtly healthy young mice (Fig 2B, +/ b). There was a
strong correlation between the degree of anemia and splenomegaly, in
that mice with the most severe anemia showed the most marked
splenomegaly (see below).
We next analyzed peripheral blood taken from 14 GATA-1.05
heterozygous mutant mice as well as 6 wild-type female litter mates. The mutant mice displayed varying degrees of anemia and
thrombocytopenia (Table 1). Whereas the severity of
anemia varied enormously among heterozygous mutant animals, the
platelet counts were consistently lower in the GATA-1.05
heterozygous mice than in wild-type litter mates (Table 1). Upon
necropsy, mice with the lowest hematocrits (hematocrit, 25) uniformly
displayed the most pronounced splenomegaly (Fig 2B). These data suggest
that the cause of death in GATA-1.05 heterozygous mice is
probably due to the anemia and splenomegaly.
Massive accumulation of proerythroblasts and megakaryocytes in the
spleens of heterozygous mutant mice.
Histological examination of the grossly enlarged spleen (of sample no.
12 in Table 1) showed that the architecture of the spleen was severely
affected (Fig 3A; compare C
and E). Importantly, proerythroblasts had massively infiltrated the
spleen (Fig 3B). Because of proerythroblasts accumulation, there was
almost no white pulp and only residual visible red pulp (see arrows).
Because the accumulating erythroid cells are immature, whereas in human myeloproliferative disorders the number of mature cells are aberrantly high, these two disorders are different in this respect.
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| Fig 3.
Histological appearance of the spleen from the
GATA-1.05 heterozygous mutant and wild-type female mice.
Histological sections of the spleens from GATA-1.05
heterozygous female mice with severe splenomegaly (A and B;
GATA-1.05 heterozygous mouse no. 12 in Table 1) or mild
splenomegaly (C and D; heterozygous animal no. 3 in Table 1) are shown.
Red pulp (R), white pulp (W), and megakaryocytes (Meg) are indicated.
The histological sections of wild-type female mouse spleen (E and F;
corresponding to wild-type no. 1 in Table 1) are also shown. Tissues
were stained with hematoxyline and eosin. Original magnifications × 20 (A, C, and E) or × 80 (B, D, and F).
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In one of the slightly enlarged spleens (from heterozygote no. 3 in
Table 1), the overall architecture of the spleen was generally
preserved, but the mass of red pulp was less prominent (Fig 3C) than
that in the wild-type spleen (Fig 3E). Furthermore, the residual red
pulp was filled with proerythroblasts (Fig 3D). The megakaryocyte count
was also significantly higher (Fig 3D and Table 1) when compared with
the number in the spleens of wild-type littermates (Fig 3F). This is in
contrast to the observation that the overall number of megakaryocytes
was somewhat diminished in the enormously enlarged spleens of the
animals that had died spontaneously (Fig 3B).
To assess functional properties of these accumulated megakaryocytes,
acetylcholinesterase and glycoprotein IIb expression was examined in
the megakaryocytes in the normal and abnormal spleens. Figure 4A and B
shows the results of acetylcholinesterase staining in a spleen of a
wild-type female, whereas Fig 4C and D shows the comparable staining
patterns for the GATA-1.05 heterozygous mouse spleen. The
number of megakaryocytes appeared to be greater in the heterozygous
mutant mouse spleen, and they were uniformly positive for
acetylcholinesterase staining. These megakaryocytes were also positive
for glycophorin IIb and displayed normal histological morphology (Fig
4E and F). Although these data demonstrated that the megakaryocytes in
the spleens of GATA-1.05 heterozygous mutants had progressed
quite far in megakaryopoiesis, the terminal steps leading to platelet
formation still seem to be impaired in these cells.
GATA-1-negative, neomycin phosphotransferase-positive cells
accumulate in the spleens of GATA-1.05 heterozygous mutant
mice.
To determine the origin of the cells that accumulate in the spleens of
GATA-1.05 heterozygous mutant mice, we performed immunostaining with an anti-GATA-1 antibody on spleen sections of the mutant and
wild-type mice. As shown in Fig 5A,
numerous cells in the red pulp of a wild-type spleen were stained brown
by the anti-GATA-1 antibody; proerythroblasts and megakaryocytes were
shown to express abundant GATA-1 (Fig 5B). In contrast, both the red
pulp and white pulp were virtually nonexistent in the enlarged spleen
of the heterozygous mutant mouse (Fig 5C). The proerythroblasts and
megakaryocytes that had accumulated in these enlarged spleens did not
immunoreact with the anti-GATA-1 antibody (Fig 5C); however, a few
GATA-1-positive erythroid cells were observed in these same sections
in remnants of the red pulp (Fig 5D). These results suggested that the
erythroid and megakaryocytic cells that have proliferated and
accumulated in the grossly enlarged mutant spleens are derived from
parental cells containing an inactivated GATA-1 allele.
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| Fig 5.
Anti-GATA-1 antibody staining of spleens. Spleens
isolated from either a heterozygous mutant female mouse with marked
splenomegaly (C and D) or a wild-type mouse (A and B). Many cells in
the red pulp (R) of the spleen of the wild-type mouse were clearly
GATA-1 positive. Note that both proerythroblasts and megakaryocytes
were GATA-1 positive. In contrast, accumulating proerythroblasts and megakaryocytes did not stain for GATA-1 in the spleens of heterozygous mutant female mice (C and D). R, red pulp; W, white pulp; Meg, megakaryocytes. Arrows stand for normal GATA-1-positive cells. Original magnifications × 10 (A and C) and × 80 (B and D).
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| Fig 7.
Histological examination of the liver, spleen, and
peripheral blood from heterozygous mutant female mice. Proerythroblasts and megakaryocytes accumulate in the sinus of the liver in
GATA-1.05 mutant females (arrow in A). (B) is a higher
magnification of (A) that contains the region pointed to by the arrow.
Original magnifications × 10 (A) and × 80 (B). Mitotic figures were
frequently seen in accumulated proerythroblasts in the spleen (arrows
in C; original magnification × 200). Proerythroblasts were also
observed in the peripheral blood of heterozygous mutant female mice
with severe anemia and marked splenomegaly (D; original magnification × 200). The sections (A) through (C) were stained with hematoxylin and eosin reagent, whereas the blood smear was stained with
Wright-Giemsa reagent (D).
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To test this hypothesis, the transcription level of neomycin
phosphotransferase (Neo) gene as well as GATA-1 gene, initiating from
both testis- and erythroid-specific promoters, were examined by RT-PCR
analysis. Because the Neo gene was knocked-in to the GATA-1 locus, we
predicted that the presence of Neo mRNA should be detectable in cells
in which the GATA-1.05 allele is active, even in the absence of
Neo selection. The analysis shown in Fig 6 verified this
hypothesis and showed that the level of Neo gene expression increased
significantly in the GATA-1.05 heterozygous mouse spleens. The
Neo expression level correlates well with the severity of splenomegaly
and bears an inverse relationship to the expression of GATA-1 from the
erythroid (IE) first exon (E-GATA-1, Fig 6). Interestingly, there is a
reciprocal increase in GATA-1 expression from the IT exon (Fig 6,
T-GATA-1). These data further support the conclusion that the mutant
GATA-1 allele is active in the cells that accumulate in the enlarged
spleens of the GATA-1.05 mutant
animals.
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| Fig 6.
RNA analysis of wild-type and heterozygous mutant female
spleens. Expression of GATA-1 from either the IE promoter (E-GATA-1) or
the IT promoter (T-GATA-1) was examined after 35 cycles of amplification using 5 primers specific for either first exon and
a 3 primer used in common in the GATA-1 third exon. Expression of neomycin phosphotransferase was also examined after 35 cycles of
amplification. G6PD was used as the internal control and analyzed after
27 cycles of amplification. The numbers indicate individual animals.
Lanes 1 and 2, samples isolated from wild-type spleens; lanes 3 and 4, heterozygous mutant female spleens in animals displaying mild
splenomegaly; lanes 5 and 6, heterozygous mutant female spleens having
marked splenomegaly. +, positive control; , no template added; M,
marker lane.
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Ectopic liver hematopoiesis in adult GATA-1.05 heterozygous
mutant mice.
The data presented thus far have shown that the cells that proliferate
and accumulate in the spleens of GATA-1.05 mutant animals are
hematopoietic cells and that they are deficient in GATA-1 synthesis.
Erythroid differentiation in the GATA-1.05 heterozygous mutant
cells bearing an active GATA-1.05 allele (and an inactive wild-type allele) is arrested at the proerythroblast stage. These data
imply that the GATA-1.05 heterozygous mutant may serve as a
mouse model for the human disorder, myelodysplastic syndromes (MDS).
MDS is characterized (among other hallmarks) by abnormal accumulation
of immature cells in the enlarged spleens of affected individuals, as
well as ectopic hematopoiesis. The notion that the GATA-1.05
mutant mice might be a good model for the human MDS is further
supported by the observation that the heterozygous mutant adult mice
that display marked splenomegaly also have ectopic hematopoiesis in the
liver, where we found erythroblast-like cells as well as megakaryocytes
in the hepatic sinuses (Fig 7A and B). Some hepatocytes were also found
to be compressed simply by accumulated hematopoietic cells. In the
spleen sections from some heterozygous mutant mice that were found
dead, mitotic figures were also often observed (Fig 7C). In addition,
high numbers of proerythroblasts were found in the peripheral blood of
mice with severe anemia (Fig 7D). These observations suggest that the
accumulation of GATA-1-deficient cells leads to a preleukemic state
and, therefore, that this mutation has the potential to serve as a
useful animal model for human MDS.
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DISCUSSION |
In this study, we have analyzed the functional contributions of GATA-1
to the growth and differentiation processes of definitive hematopoietic
cells in heterozygous GATA-1 knock-down mutant mice. We took advantage
of the facts that the murine GATA-1 gene is localized on the X
chromosome and that one of the two X chromosomes is inactivated
randomly in heterozygous females. The loss of GATA-1 function in this
mutant resulted in the stimulation of growth and the arrest of the
differentiation in hematopoietic cells, which became manifest as severe
anemia, thrombocytopenia, and splenomegaly, thus resulting in a
foreshortened life span. The data demonstrate that GATA-1 is necessary
for the terminal differentiation of definitive erythroid and
megakaryocytic lineage cells in vivo.
GATA-1 has long been thought, and more recently demonstrated, to be a
key regulator of erythroid lineage maturation. For instance, we and
others recently demonstrated that maturation of primitive erythroid
cells is arrested at the proerythroblast stage.14,15 Compared with the unequivocal proof supporting the role of GATA-1 in
primitive hematopoiesis, the evidence of GATA-1 in definitive hematopoiesis has been quite limited. In this regard, Fujiwara et
al15 recently reported that the majority of mice
heterozygous for a GATA-1-null allele are born anemic, but they
recover during the neonatal period. They speculated that this recovery
is presumably due to in vivo selection of progenitor clones with an
active normal allele. We have observed the same phenomenon in mice
bearing the knock-down GATA-1.05 allele (data not shown). In
addition, we report here that mice heterozygous for the
GATA-1.05 allele have a significantly shorter life span
and splenomegaly. The observation of late onset splenomegaly in the
GATA-1.05 heterozygous mutant mice was an intriguing surprise.
We demonstrated that the cells that had proliferated and accumulated in
these mutant animals are of the erythroid and megakaryocytic lineages.
The proliferative cell population was shown to contain an active
GATA-1.05 allele (and an inactive wild-type allele) and
undetectable amounts of GATA-1 protein. In summary, these data
indicated that, in the absence of GATA-1, hematopoietic progenitors
either have, or gradually acquire, a growth advantage over their normal
counterparts some time during postnatal development.
Forced expression of GATA-1 protein in avian erythroid progenitor cells
was reported to accelerate erythroid cell differentiation with
concomitant suppression of proliferation,20 whereas
constitutive expression of GATA-1 was reported to interfere with normal
cell-cycle progression.21 These reports and our present
data lead us to hypothesize that the lack of GATA-1 stimulates
proliferation and suppresses differentiation in hematopoietic cells. In
this regard, an in vitro ES cell differentiation system developed by
Nakano et al22 may serve as an additional useful
experimental tool for detailed assessment of the erythroid
differentiation process. Our preliminary analysis of
GATA-1.05/Y ES cells14 using this method has showed
that the GATA-1.05 mutation directly affects the growth rate of
proerythroblasts and megakaryocytes (Suwabe et al, submitted for
publication). Because the only resident feedback mechanism
that exists in the in vitro ES cell differentiation system is provided
by the support cells, which may represent the bone marrow stroma
environment (see below), these data further support the hypothesis that
GATA-1 acts simultaneously to stimulate hematopoietic cell
differentiation and to suppress proliferation.
In contrast to our present finding, Weiss et al12 reported
that GATA-1 proerythroblasts simply apoptosed in
vitro. We think that this may be accounted for by the difference in in
vivo and in vitro experimental systems. A similar discrepancy has been
seen previously, in that primitive erythrocytes could not be generated
from GATA-1-null ES cells under their in vitro differentiation
condition. We14 and others15 have found that
primitive erythrocytes are generated, but not terminally
differentiated, under the GATA-1 knock-down14 or knock-out
condition15 in vivo.
Expression of GATA-1 from the IE promoter was found to be significantly
diminished in abnormally abundant hematopoietic cells in the spleen of
the GATA-1.05 mutant females. In contrast, GATA-1 expression
from the IT promoter concomitantly increased in the anemic mice that
displayed splenomegaly. Moroni et al23 recently reported
that, when purified hematopoietic progenitors are stimulated by
erythropoietin, GATA-1 expression from the IT promoter was upregulated.23 Therefore, the more abundant expression of
GATA-1 arising from the IT promoter in the heterozygous mutant mice
could be in response to increased levels of erythropoietin in these animals. The data therefore suggest that the expression of GATA-1 from
the IT promoter may be under regulatory feedback control in a
homeostatic circuit involving the growth control elicited by
erythropoietin. However, the amount of GATA-1 from the IT promoter is
probably below the sensitivity of histochemical detection, because the
normal expression level of GATA-1 from the IT promoter in hematopoietic
cells is only about 5% of the total GATA-1 expression in the spleen.
Despite the abnormal accumulation of megakaryocytes in the mutant
spleens, a severe and consistent thrombocytopenia was observed in the
GATA-1.05 heterozygous female mice. We therefore concluded that
GATA-1 may also be important for the terminal differentiation of
megakaryocytes. Consistent with this observation, Pevny et al13 previously reported that GATA-1-negative
megakaryocytes were abnormally abundant in chimeric fetal livers,
suggesting an alteration in the kinetics of their formation or
turnover. Furthermore, Shivdasani et al24 have recently
demonstrated that GATA-1 plays a critical role in megakaryocyte growth
regulation and platelet biogenesis in vivo using a lineage-specific
knock-out strategy. All of these results argue that GATA-1 is necessary for terminal differentiation of megakaryocytes.
Megakaryocytes normally express GATA-1, and several genes expressed
specifically in this lineage appear to be under the regulation of
GATA-1 or other GATA family transcription factors. It has been reported
that the glycoprotein IIb gene is directly regulated by
GATA-1.25 However, we detected the glycoprotein IIb protein in megakaryocytes that lack GATA-1. This suggests either that GATA-2,
which is coexpressed in this cell lineage, could functionally substitute for GATA-1 or that the low level of GATA-1 present in the
GATA-1.05 mutant megakaryocytes may be adequate to execute most, but not all aspects, of megakaryocytic differentiation. Regardless of the mechanism used, the results presented here clearly indicate that GATA-2 cannot fully compensate for GATA-1 function in
megakaryopoiesis. Thus, the identification of bona fide target genes of
GATA-1 remains an important hurdle to extending our understanding of
the role of GATA-1 in megakaryocyte differentiation.
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FOOTNOTES |
Submitted October 16, 1997;
accepted March 6, 1998.
Supported by the Grants-in-Aid from the Ministry of Education, Science,
Sports and Culture, Japanese Society for Promotion of Sciences
(RFTF96I00202) and the Ciba-Geigy Foundation (Japan).
Address reprint requests to Masayuki Yamamoto, MD, PhD, Center for TARA
and Institute of Basic Medical Institute, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305, Japan; e-mail: masiya{at}igaku.md.tsukuba.ac.jp
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
The authors thank Drs J.D. Engel, N. Kajiwara, N. Kasai, H. Nakauchi,
K.-C. Lim, H. Sugiyama, N. Suzuki, N. Takasawa, and K. Yagami for help
and discussion.
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Abstract
Introduction
Abstract