(Received for publication, January 24, 1997, and in revised form, February 17, 1997)
From the To elucidate the in vivo function of
GATA-1 during hematopoiesis, we specifically disrupted the erythroid
promoter of the GATA-1 gene in embryonic stem cells and generated germ
line chimeras. Male offspring of chimeras bearing the targeted mutation
were found to die by 12.5 days post coitus due to severe anemia while heterozygous females displayed characteristics ranging from severe anemia to normal erythropoiesis. When female heterozygotes were crossed
with transgenic males carrying a reporter gene, which specifically
marks primitive erythroid progenitors, massive accumulation of
undifferentiated erythroid cells were observed in the yolk sacs of the
GATA-1-mutant embryos, demonstrating that GATA-1 is required for the
terminal differentiation of primitive erythroid cells in
vivo.
The transcription factor GATA-1 (1-3) is expressed in erythroid,
megakaryocytic, and mast cells (4, 5) as well as in the Sertoli cells
of the testis (6). GATA-1 is encoded by the X chromosome (7), and two
alternative promoters, which direct GATA-1 expression in distinct cell
lineages, have been identified. The 5 Analysis of GATA-1-null embryonic stem
(ES)1 cells generated by homologous
recombination has revealed that the mutant cells do not contribute to
the mature erythroid population in chimeric animals (9). These ES cells
also failed to differentiate into mature erythroid cells in
vitro (10-12), suggesting that GATA-1 is required for the
erythroid differentiation of hematopoietic progenitor cells. However,
previous attempts failed to produce germ line transmission of the
GATA-1-null allele. Thus, the in vivo role of GATA-1 in
erythropoiesis remains to be resolved.
In this study, we enfeebled the GATA-1 gene through a disruption of its
hematopoietic promoter in ES cells and generated germ line chimeras to
directly address the in vivo effects of the targeted mutation. Our results demonstrate that GATA-1 is required for the
terminal differentiation of primitive erythroid cells in
vivo, and this work reveals for the first time the consequence of
GATA-1 function in primitive erythroid progenitors during
development.
Genomic clones of the GATA-1 locus
were isolated from a mouse 129 SVJ RNA from 9.5 days post
coitus (dpc) embryos was isolated by single-step RNA extraction system
(RNAzol, Tel-Test). First strand cDNA was synthesized using
Superscript reverse transcriptase (Life Technologies, Inc.) at 37 °C
for 1 h, and 1 µl of this 20-µl reaction mixture was used in
PCR reactions. Reaction products were analyzed on 2% agarose gels.
Sequences of the primers used and the expected sizes of PCR products
are as follows: for erythroid-specific GATA-1, 5 Newborn
mice were fixed in 10% buffered formalin and embedded in paraffin.
Sections were stained with hematoxylin and eosin for histological
examination. Embryos were fixed after being dissected free of maternal
tissues. Fetal liver cells were inoculated
into a methyl cellulose culture in the presence of erythropoietin (2 units/ml) (18). After 2 days, the number of colonies formed were
counted, colony forming unit-erythroid (CFU-E). Cells were also assayed
for the ability to form granulocyte colonies, colony forming
unit-granulocyte/macrophage (CFU-GM) in a semi-solid culture
supplemented with granulocyte/macrophage-colony stimulating factor,
IL-3, and IL-6.
We prepared
a positive/negative selection targeting vector to introduce a neo
cassette into the intergenic region between an important regulatory
region of the erythroid promoter and the mRNA cap site. GATA factor
binding sites located approximately 620 bp 5
This targeting construct was introduced by electroporation into ES
cells, and 20 independent G418-resistant, PCR-positive clones were
identified. Southern blot analysis using internal gene-specific and neo
probes confirmed the homologous recombination events (Fig. 1,
B and C). Three mutant ES cell lines were used for the generation of chimeric offspring. All the ES cell lines gave
rise to chimeras. We crossed these chimeras with normal Balb/c mice and
one line (No. 78) gave stable germ line transmission. All F1 generation
mice from the chimera mouse were found to be of ES cell origin, and the
number of litters and sex ratio were in normal range.
F1 progeny were
examined by Southern blot and PCR analyses to confirm the presence of
the mutated GATA-1 allele. Since the GATA-1 gene is located on the X
chromosome, all heterozygous F1 pups were female. These heterozygotes
were found to display varying degrees of anemia. Fig.
2A shows GATA-1 heterozygous embryos; their
appearance ranges from normal (left) to severely anemic (right). Concordantly, when we analyzed the livers of two
heterozygous newborn littermates (panel B), we found that
one liver contained a normal number of hematopoietic cells
(left), whereas the other displayed a marked reduction in
the number of hematopoietic cells (right). These
observations indicated that disruption of the GATA-1 gene erythroid
promoter seems to cause severe anemia in vivo and that the
effect of the knock-down strategy was as anticipated. The heterogeneity
of the phenotypes observed in the female heterozygotes may be the
result of selective inactivation of the X chromosome (22).
The heterozygous females were mated with wild-type
males to observe the phenotypic effects on mutant males. Surprisingly, no mutant males were observed among the live offspring, and
heterozygous female offspring displayed the same varying degrees of
anemia that we observed among the F1s. To determine the nature and
timing of the presumptive embryonic lethality of the targeted males, we
collected and genotyped embryos ranging from 8.5 to 14.5 dpc. A total
of 112 pups from 13 litters were analyzed. At 9.5 dpc, mutant male
embryos were present in the expected ratio, but already visible defects
could be discerned. The yolk sacs of mutant males were noticeably pale
and almost no blood vessels were present (Fig.
3A, right). The mutant males (Fig.
3B, right) could be easily distinguished from
wild-type (left) or heterozygous female embryos (data not
shown) by their distinctive pallor and small size. Embryonic lethality
was first detected at 11.5 dpc, and no live mutant male embryos were
found by 12.5 dpc.
The expression levels of erythroid-specific GATA-1 mRNA were
analyzed by RT-PCR using RNA samples obtained from 9.5-dpc embryos. Densitometric analysis of the RT-PCR data revealed that the expression of GATA-1 from the IE promoter in the mutant male embryos was less than
5% of the level present in wild-type embryos (Fig.
4A), therefore we will refer to this targeted
mutation as the GATA-1.05 mutant allele. These data show that the
expression of the GATA-1 gene from the IE promoter is severely
impaired, but not extinguished, by the promoter disruption. The
phenotype and timing of death in the male mutant embryos led us to
conclude that the cause of in utero lethality is due to
impaired primitive erythropoiesis.
We also analyzed the expression of GATA-1 from the testis-specific
promoter (IT promoter) in these embryos. The expression of testis-type
GATA-1 mRNA in the mutant embryos was almost identical as that seen
in the wild-type embryos (Fig. 4B), indicating that activity
of the testis promoter was left intact in our knock-down mice.
We examined the hematopoietic phenotype in the livers of
mutant male embryos. Morphological examination revealed that size of
the liver rudiments of mutant male embryos was markedly reduced and
that the color was pale in comparison to the livers of wild-type embryos (data not shown). Microscopic analysis revealed that, whereas
many erythroid cells should be present in the liver rudiment at 11.5 dpc, virtually none could be detected in the livers of mutant embryos
(compare Fig. 5, panels A and B).
This seems to reflect the absence of yolk sac hematopoiesis.
GATA-1-positive cells were also absent in liver sections from 11.5-dpc
GATA-1.05 mutant male (compare panels C and
D).
We also performed in vitro colony assays with these same
fetal liver cells. In comparison with wild-type littermates, the GATA-1.05 male mutant livers did not generate any CFU-E at 10.5 dpc
(data not shown), and only a small number of CFU-E were recovered at
11.5 dpc (Table I). In contrast, fetal liver cells from
either wild-type or mutant embryos produced similar numbers of CFU-GM colonies (Table II). These data indicate that only a
small number of erythroid progenitors exist in the liver of the mutant
embryos, while the non-erythroid hematopoietic lineages are unaffected by the mutation. The number of CFU-E formed from fetal liver cells of
female heterozygous mutants (GATA-1(+/1.05)) accordingly displayed wide
variability, again consistent with the hypothesis that the X chromosome
is selectively inactivated in hematopoietic progenitor cells.
Table I.
Results of the fetal liver CFU-E assays
Table II.
Results of the fetal liver CFU-GM assays
Center for Tsukuba Advanced Research
Alliance and Institute of Basic Medical Sciences, University of
Tsukuba, Tennodai, Tsukuba 305, Japan, the § Department of
Biochemistry,
Department
of Molecular Genetics, National Institute of Neuroscience,
Ogawa-higashi, Kodaira 187, Japan
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
promoter specifies expression in
Sertoli cells while a downstream promoter located between the first
testis-specific exon and the common coding exons of the gene directs
the expression of GATA-1 in erythroid cells (8).
GATA-1 Gene Targeting
Fix II library (Stratagene). A
5.1-kilobase 5
-fragment and 2.7-kilobase 3
-fragment, which contains
the erythroid-specific first exon (IE exon), were cloned into a vector
containing a neomycin-resistance gene (neo) cassette to generate the
targeting construct. The resultant construct was linearized with
HindIII and electroporated into E14 cells as described (13).
Clones were selected in G418 (300 µg/ml), expanded, and analyzed by
PCR. Homologous recombination was observed at a frequency of ~1/10
G418-resistant clones. Chimeras were generated by injection of C57 BL/6
blastocysts as described previously. Pups and embryos were genotyped by
PCR and/or Southern blotting analyses. We determined the sex of these
embryos by using PCR amplification of Y chromosome-specific
zfy-1 gene (14).
-TAAGGTGGCTGAATCCTCTGCATC and 3
-CCGTCTTCAAGGTGTCCAAGAACGT (expected product 483 bp); and for testis-specific GATA-1,
5
-CGTGAAGCGAGACCATCGTC and the 3
-primer used for the
erythroid-specific primer set (expected product 529 bp). Primers for
glucose-6-phosphate dehydrogenase were synthesized as described
previously (expected product 162 bp; Ref. 15).
-Galactosidase (LacZ) activity was detected as described
previously (16). After
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal)
staining, embryos were photographed before embedding in OCT compound
(Sakura Finetechnical) and freezing. Cryosections were processed for
immunohistochemistry with anti-murine embryonic hemoglobin sera (17),
which was kindly provided by Dr. Tadao Atsumi.
Targeted Disruption of the Mouse GATA-1 Promoter
to the IE exon (Fig.
1A) have been shown to positively regulate
transcription from the IE promoter (19). The insertion of neo was
designed to separate the GATA motifs from the IE exon in the hope that
the mutation would negatively affect the activity of the IE promoter.
In addition, the positive/negative selection targeting vector was
designed to transcribe neo in the same orientation as the GATA-1 gene
so that the strong promoter directing neo expression might further
weaken the activity of the IE promoter through competition (20, 21).
Our expectation is that the activity of the IE promoter will be
diminished by the combinatorial effects of these two mechanisms, resulting in a specific `knock-down' (rather than a `knock-out') of
the erythroid-specific expression of the GATA-1 gene, and possibly lead
to a germ line-transmissible targeted mutation.
Fig. 1.
Targeted disruption of the mouse GATA-1
promoter. A, targeting strategy. Solid boxes
depict exons (IE and IT denote the
erythroid- and testis-specific first exons, respectively). The
MC-1-neomycin (Neo) gene (large open box) was
inserted between the double GATA sequences (round open
circle) and IE exon. Probe 1 and Neo probe were used in Southern
blotting analysis to detect homologous recombination. Small open
box (DT) indicates the diphtheria toxin A gene cassette
(26). Southern blot analyses of SacI-digested chromosomal
DNA isolated from wild-type (lanes 1 and 3) or
mutant (lane 2) ES cell clones hybridized with probe 1 (B) or neo probe (C).
[View Larger Version of this Image (26K GIF file)]
Fig. 2.
Variation in the severity of phenotypes
exhibited in heterozygous female embryos. A, two female
heterozygous 15-dpc embryos are shown. The pale color of the
right embryo contrasts sharply with the normal coloring of
its sibling. B, histological examination of livers of
heterozygous neonates. Few hematopoietic cells are observed in the
section on the right as compared with the relatively normal
number of cells in the left section.
[View Larger Version of this Image (103K GIF file)]
Fig. 3.
Phenotypes of GATA-1.05 mutant male
embryos. Visualization of the highly vascularized wild-type yolk
sac (panel A, left) and embryo (panel
B, left) versus anemic yolk sac and embryo of a mutant male embryo (right) from the same litter at 9.5 dpc of embryogenesis.
[View Larger Version of this Image (99K GIF file)]
Fig. 4.
RT-PCR analysis of total RNA isolated from
mutant male, heterozygous female, and wild-type 9.5-dpc embryos.
A, expression of GATA-1 from the IE promoter
(E-GATA-1) was examined after 25, 30, and 35 cycles of amplification. Glucose-6-phosphate dehydrogenase was
used as the internal control and analyzed after 22, 27, and 32 cycles
of amplification. The numbers indicate individual embryos. Lanes 1-3 are mutant male embryos, lanes 4-6
are heterozygous female embryos, and lanes 7-9 are
wild-type embryos. B, expression of GATA-1 from IT promoter
(T-GATA-1) were examined after 35 cycles of amplification. The same
embryo samples used in panel A were used to detect the IT
promoter expression. Lanes are arranged the same way as in
panel A.
[View Larger Version of this Image (58K GIF file)]
Fig. 5.
Decreased number of erythroid cells in mutant
fetal livers. Hematoxylin and eosin staining of the liver sections
of 11.5-dpc wild-type (A) and GATA-1.05 mutant male
(B) embryos are shown. The arrowhead indicates a
cluster of hemoglobinized cells. Anti-GATA-1 antibody staining of a
12.5-dpc wild-type embryonic liver section (C) and an
11.5-dpc GATA-1.05 male mutant (D) are also shown. The
arrowhead indicates a GATA-1-positive cell.
[View Larger Version of this Image (120K GIF file)]
Genotype
Wild
type
+/
female
/Y male
Embryo number
1
52,600 ± 1,350
2
56,200
± 1,810
3
49,120 ± 6,350
4
2,590
± 168
5
110,370 ± 611
6
33,410 ± 767
7
18,500 ± 180
8
88,760 ± 3,276
9
109,120 ± 1,210
10
34,020 ± 2,814
11
1,360 ± 164
12
69,750 ± 3,726
13
59,044 ± 4,303
14
72,027 ± 5,819
15
11,175 ± 892
Genotype
Wild type
+/
female
/Y male
Embryo number
21
207 ± 23
22
1056 ± 102
23
1476 ± 36
24
378 ± 6
25
276 ± 138
26
700 ± 161
28
320 ± 40
To investigate how primitive hematopoiesis is affected by the lack of GATA-1, we took advantage of a GATA-1 promoter-LacZ transgene, which is transcriptionally active in primitive but not definitive erythroid cells, to specifically mark the primitive erythroid lineage (23). Female mice heterozygous for the GATA-1.05 mutation were crossed with transgenic mice homozygous for the LacZ reporter transgene to generate LacZ-positive embryos harboring the GATA-1.05 mutation (compound mutation designated GATA-1.05::LacZ).
In 8.0-dpc embryos examined by whole mount X-gal staining, no
significant difference in either the abundance or distribution of
stained erythroid cells was observed between GATA-1.05::LacZ mutant embryos and LacZ-only littermates (data not shown). By 9.5 dpc,
however, the number of blue cells detected in the
GATA-1.05::LacZ embryos was far greater than that seen in
littermates carrying only the reporter transgene (compare panels
A and B, Fig. 6). Detailed examination
revealed that the absolute number of hematopoietic cells found in the
yolk sac, aortic sac, and atrial chamber of the heart was similar in
both wild-type and GATA-1.05 mutant embryos. The significant difference
was that only a small percent of the hematopoietic cells in the atrial
chamber were positive for staining in the LacZ control embryos
(panel C), compared with the blue staining seen in virtually
all of the hematopoietic cells in the GATA-1.05::LacZ male
embryos (panel D). These blue GATA-1-positive cells express
only a limited amount, if any, of embryonic hemoglobin as visualized by
an embryonic hemoglobin anti-sera. It should be noted that, consistent
with the whole mount analysis described above, virtually all
hematopoietic cells in the yolk sac blood islands of 8.5-dpc LacZ
transgenic mouse embryos were blue regardless of whether they carried
the GATA-1.05 or wild-type allele (data not shown), confirming the
specific expression of the reporter GATA-1/LacZ transgene in
undifferentiated primitive hematopoietic progenitors. Therefore, in the
GATA-1.05 mutant background, it seems that primitive hematopoietic
progenitors develop to the GATA-1-positive stage but subsequently
cannot differentiate into normal hemoglobinized cells.
We show here that a modified gene targeting strategy can be successfully employed to dissect the complex roles played by GATA-1 in vivo. This strategy consisted of two components. The first is promoter-specific impairment (knock-down) of transcription factor gene activity, and the second is the lineage- and stage-specific marking of the lineage of target cells that specifically require the function of the transcription factor. The combination of these two approaches enabled us to identify the precise stage of maturation arrest within the primitive erythroid lineage that resulted as a direct consequence of the lowered levels of GATA-1. The in vivo approach described here may be applied to the analysis of any gene of interest with well characterized regulatory regions.
We are able to detect the accumulation of primitive erythroid progenitors in the yolk sac of the GATA-1-mutant embryos, demonstrating that GATA-1 is necessary for the differentiation, but not the formation, of primitive erythroid progenitors. These results are especially intriguing and contrast with the conclusions of a previous report (24). Using in vitro differentiation of GATA-1-null ES cells, Weiss et al. (11) failed to detect primitive erythroid precursors, leading them to conclude that GATA-1 is essential at the earliest stage of primitive erythropoiesis definable by then current methods. In contrast, we observed numerous primitive erythroid progenitors in the yolk sacs and atrial chambers of GATA-1.05 mutant embryos, and the erythroid enhancer of GATA-1 seems active in the progenitor cells. Two possibilities could account for this discrepancy. First, it is possible that a cell lacking GATA-1 may be able to survive in vivo but not under in vitro conditions (24). Alternatively, the residual 5% activity displayed by GATA-1 in the 1.05 mutant allele might be sufficient for cell survival but not for promoting terminal differentiation. We favor the former hypothesis since the knock-down effect on erythropoiesis seems to be clonal, and not uniform, in the erythroid cells (some weakly hemoglobinized cells in the yolk sac of the mutant embryos are routinely detected).
In this regard, it should be noted that Fujiwara et al. (25)
recently reported the germ line transmission of a GATA-1-null allele.
In that study, the second exon of the GATA-1 gene was disrupted in ES
cells, as was the method employed in several previous trials (9-12),
and thus the reasons for the failure to generate germ line chimeras
using the original GATA-1-disrupted ES cells prepared for previous
studies remains unclear. In agreement with the present analysis, male
mice bearing the GATA-1-null allele (i.e. /Y genotype)
died by 11.5 dpc of embryogenesis. The coincidence of the null and
knock-down mutant phenotypes suggests that the knock-down strategy
resulted, as anticipated, in enfeebling the gene to such an extent that
its final activity was very close to that of a null mutation, while
analysis of the Sertoli GATA-1 transcript indicates that its synthesis,
as also anticipated, is unaffected by the GATA-1.05 mutation. Fujiwara
et al. (25) also found accumulation of embryonic erythroid
cells in GATA-1-null embryos and suggested that the inability to detect
embryonic erythroid precursors in prior in vitro experiments
reflected a limitation of cell culture assays. Finally, they suggested
that the accumulated cells in the yolk sac of the null mutant mouse
were arrested at the proerythroblast stage based on morphological
examination. In contrast, the present study provides direct evidence
for the accumulation of primitive erythroid progenitors in the yolk sac blood islands through an intercross experiment performed by breeding the GATA-1.05 mutant allele to a separate line of mice bearing a
transgene that specifically marked the primitive erythroid lineage. As
additional evidence, we were able to unequivocally identify the
arrested cells as primitive erythroid progenitors using both anti-GATA-1 and anti-embryonic hemoglobin antibodies.
In addition to the primitive erythroid lineage analysis, preliminary studies on the effect of GATA-1 mutation on fetal liver (definitive) erythropoiesis in vivo are presented here for the first time, and we also found this developmental process to be severely affected by the knock-down mutation. The mutant fetal livers were observed to contain only a small number of CFU-E. Since we detected comparable numbers of CFU-GM in either GATA-1(+) or GATA-1.05 mutant mice, the GATA-1 deficiency appears to specifically promote the growth, or prevent apoptosis, in definitive erythroid progenitors. However, we cannot completely rule out the indirect physiological influence (e.g. through hypoxia) that may result from the lack of primitive erythropoiesis. Gene rescue and/or selective inactivation experiments will be necessary to further refine the specific roles played by GATA-1 in definitive hematopoiesis in vivo.
We thank Drs. R. Yu (Nara Advanced Institute of Science and Technology), T. Atsumi (RIKEN), K. Igarashi (Center for Tsukuba Advanced Research Alliance), K. Araki (Kumamoto University), E. Ito (Hirosaki University), Y-K. Nabeshima (National Institute of Neuroscience), N. Kasai, T. Ono, and M. Obinata (Tohoku University) for help and discussion.