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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2771-2779
RAPID COMMUNICATION
Mice Homozygous for a Truncated Form of CREB-Binding Protein Exhibit
Defects in Hematopoiesis and Vasculo-angiogenesis
By
Yuichi Oike,
Nobuyuki Takakura,
Akira Hata,
Tadashi Kaname,
Miwa Akizuki,
Yuji Yamaguchi,
Hirofumi Yasue,
Kimi Araki,
Ken-ichi Yamamura, and
Toshio Suda
From the Department of Developmental Genetics, the Department of Cell
Differentiation, Institute of Molecular Embryology and Genetics and the
Department of Cardiovascular Medicine, Kumamoto University School of
Medicine, Kumamoto; and the Department of Public Health, Asahikawa
Medical College, Asahikawa, Japan.
 |
ABSTRACT |
CREB-binding protein (CBP) and the closely related adenovirus
E1A-associated 300-kD protein (p300) function as coactivators of
transcription factors such as CREB, c-Fos, c-Jun, c-Myb, and several
nuclear receptors. To study the roles of CBP in embryonic development,
we generated CBP homozygous mutant mouse embryos that expressed a
truncated form of CBP protein (1-1084 out of 2441 residues). The
embryos died between embryonic days 9.5 (E9.5) and E10.5 and exhibited
a defect in neural tube closure. They appeared pale and showed
decreases in erythroid cells and colony-forming cells (CFCs) in the
yolk sac, suggesting defects in primitive hematopoiesis.
Immunohistochemistry with an anti-PECAM antibody showed a lack of
vascular network formation. Organ culture of para-aortic
splanchnopleural mesoderm (P-Sp) with stromal cells (OP9) showed an
autonomous abnormality of putative endothelial precursors, which may
induce the microenvironmental defect in hematopoiesis. In addition,
these defects were partially rescued by the addition of VEGF to this
culture. Our analyses demonstrate that CBP plays an essential role in
hematopoiesis and vasculo-angiogenesis.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
CREB-BINDING PROTEIN (CBP) and the
closely related adenovirus E1A-associated 300-kD protein (p300)
function as versatile coactivators of transcription factors such as
CREB, c-Fos, c-Jun, c-Myb, and several nuclear
receptors.1-7 Both CBP and p300 also play roles in
chromatin remodeling, signaling pathways, and basic cellular functions
such as DNA repair, cell growth, differentiation, embryonic
development, and tumor suppression.8,9 The mutations in CBP
gene were reported to be associated especially with the Rubinstein-Taybi syndrome in humans10 and
mice.11,12 The role of CBP in embryonal development is not,
however, well understood.
CBP heterozygous mutant mice are viable and fertile and show clinical
features of Rubinstein-Taybi syndrome,11,12 but the CBP
homozygous mutantation is embryonic lethal. Recently, Yao et
al13 reported that p300-deficient mice show embryonic
lethality due to poor cardiac development in the presence of normal
levels of CBP. These data indicate that physiological and biochemical functions of CBP and p300 do not fully overlap, at least during embryonic development.
During mouse embryogenesis, hematopoiesis begins in the yolk sac at
E7.5 and it shifts to the fetal liver and then to the spleen and bone
marrow.14 Hematopoiesis before formation of the fetal liver
is known as primitive hematopoiesis and is distinguished from the
adult-type definitive hematopoiesis by specific expression of an
embryonic-type of globin in nucleated erythrocytes. In the mouse
embryo, a pre-liver intraembryonic site of definitive hematopoietic activity has been identified.15 This mesodermally derived
region of the mouse embryo contains the para-aortic splanchnopleural mesoderm (P-Sp) region or dorsal aorta, genital ridge/gonads and pro/mesonephros (aorta-gonad-mesonephros, AGM) and has been shown to
harbor adult-type multipotent hematopoietic stem
cells.15-18 The development of hematopoiesis is closely
linked to that of vasculo-angiogenesis. Endothelial and hematopoietic
cells in blood islands are proposed to originate from a common
precursor, termed the hemangioblast, based on their simultaneous
emergence. To clarify the function of CBP in embryonal development, we
performed a close examination of the CBP mutant embryos with special
attention to defects in the hematopoietic and vascular system.
| |
MATERIALS AND METHODS |
Analysis of DNA and RNA.
For preparation of genomic DNA, embryonic stem (ES) cells
were lysed with sodium dodecyl sulfate (SDS) proteinase K and with phenol/chloroform, 1:1 (vol/vol) twice. The genomic DNA was
precipitated with ethanol and dissolved in 10 mmol/L Tris-HCl, pH 7.5/1
mmol/L EDTA. Six micrograms of genomic DNA was digested with
appropriate restriction enzymes, electrophoresed on a 0.9% agarose
gel, and blotted onto a nylon membrane (Boehringer Mannheim, Mannheim, Germany). Hybridization was performed using a DIG DNA Labeling and
Detection Kit (Boehringer Mannheim).
For genotyping of the embryos, we used reverse transcriptase-polymerase
chain reaction (RT-PCR) to detect fragments of the wild-type CBP and
recombinant CBP/ -geo fusion transcript. Total tissue RNA from E9.5
embryos or yolk sacs was extracted with TRIZOL (GIBCO-BRL,
Gaithersburg, MD). cDNA from total RNA was synthesized with Superscript
II Preamplification System (GIBCO-BRL). In brief, 3 µg of total RNA
was digested with RNase free-DNase, heated at 70°C for inactivation
of the enzyme, and denatured and hybridized with oligo dT primers.
First-strand cDNA was synthesized by RT, and the RNA was digested with
RNase H. The first-strand cDNA was used as the template for PCR with
forward (5'-gtctgagatgatggaagagg-3') and reverse
(5'-gacctccacagtcttgtctg-3') primers in a reaction consisting of 28 cycles of denaturation at 94°C for 60 seconds, annealing at 58°C
for 90 seconds, and extension at 72°C for 90 seconds. Amplification
of the wild-type transcript with these primers generates a 1,060-bp CBP
fragment. To detect the CBP/-geo fusion transcript, the same CBP
forward primer and a splicing acceptor (SA) reverse primer
(5'-tgctctgtcaggtacctgttgg-3') were used for PCR under conditions
described above. Amplification with this set of primers generates a
328-bp CBP/-geo fusion cDNA fragment.
Western blotting.
Homogenates from CBP+/+ and
CBP+/Mut adult mouse brain and
CBPMut/Mut embryos were prepared as previously
described.11,19 Thirty micrograms of brain or 5 µg of
whole embryo homogenate proteins were subjected to 0.1% SDS/7.5%
polyacrylamide gel electrophoresis (PAGE) as described by
Laemmli.20 Proteins were then transferred to a nitrocellulose filter (Millipore, Bedford, MA) and detected using anti-CBP (CBP-A22 or CBP-C20; Santa Cruz, Santa Cruz, CA)
antibodies21 and the ECL Detection System (Amersham,
Arlington Heights, IL).
In vitro culture of P-Sp.
P-Sp explants derived from E9.5 CBP heterozygous and homozygous mutant
embryos were cultured at 37°C in humidified 5% CO2 air
on a layer of OP9 stromal cells. OP9 cells were maintained in
 -modified minimum essential media (-MEM; GIBCO-BRL) supplemented with 20% fetal calf serum (FCS; JRH Biosciences, Renexa, KS). Explants
of E9.5 P-Sp containing a part of the omphalomesenteric artery (OA)
were cultured on OP9 stromal cells in 10% FCS and 10 5
mol/L 2-mercaptoethanol (2ME; Sigma, St Louis, MO) with or without full-length VEGF (Pepro Tech EC Ltd, London, UK). After 14 days in
culture, an anti-PECAM-1 antibody (MEC13.3, rat anti-mouse monoclonal;
Pharmingen, San Diego, CA) was used to stain vascular cells.22
To examine definitive hematopoiesis, explants of P-Sp were cultured on
OP9 cells in RPMI1640 (GIBCO-BRL) supplemented with interleukin-6
(IL-6) (20 ng/mL), IL-7 (20 ng/mL) (gifts from Dr T. Sudo, Toray
Industries Inc, Kamakura, Japan), stem cell factor (SCF)
(50 ng/mL) (a gift from Chemo-Sero-Therapeutic Research Institute,
Kumamoto, Japan), and erythropoietin (Epo) (2 U/mL) (a gift from
Snow-Brand Milk Product Co, Tochigi, Japan) at 37°C in a humidified
5% CO2 air. The in vitro colony assay was performed in
methylcellulose-containing medium as described
previously.22,23 Briefly, cells disaggregated from the
culture of P-Sp explants were obtained after 10 days, then plated in 1 mL of culture medium containing -MEM, 1.2% methylcellulose (Aldrich
Chemical Co, Milwaukee, WI), 30% FCS, 1% deionized bovine serum
albumin (BSA; Sigma), 50 mmol/L 2ME, 200 U/mL IL-3, 2 U/mL Epo, and 50 ng/mL SCF. On the seventh day of culturing, aggregates consisting of 40 or more cells were counted as a single colony.
Whole-mount immunohistochemistry.
For whole-mount immunohistochemistry embryos were fixed in 4%
paraformaldehyde at 4°C overnight. The fixed embryos were then rinsed
in phosphate-buffered saline (PBS), dehydrated in a methanol series,
and stored in 100% methanol at  80°C. The dehydrated embryos were
bleached in methnol plus 5% vol/vol hydrogen peroxide for 4 to 5 hours
at 4°C. The bleached embryos were rehydrated and blocked in PBSMT
(2% instant skim milk, 0.1% Triton X-100, PBS) for 1 hour twice. The
embryos were then incubated with 1:500 diluted anti-PECAM-1 antibody
(MEC13.3) in PBSMT at 4°C overnight. The next day, the embryos were
washed with PBSMT at 4°C five times (1 hour each) and incubated
overnight at 4°C with 1 µg/mL horseradish peroxidase-conjugated
goat anti-rat IgG in PBSMT. The next day, the embryos were washed with
PBSMT at 4°C five times (1 hour each) and in PBST (0.1% Triton
X-100, PBS) for 6 minutes three times at room temperature. Peroxidase
staining was performed by incubating embryos in 0.3 mg/mL DAB (Sigma),
0.8 mg/mL NiCl2 in PBST for 20 minutes, followed by
addition of hydrogen peroxide to a final concentration of 0.05%. The
best signal-to-background ratio was typically achieved by a 5- to
10-minute incubation. The staining reaction was stopped by rinsing in
PBST followed by postfixing in 0.1% glutaraldehyde in PBS at 4°C overnight.
| |
RESULTS |
To identify functionally unique genes in embryogenesis, we took
advantage of a random mutagenesis system using a gene-trapping strategy.24,25 First, we introduced the trapping vector,
pU-San (Fig 1A), into TT2 ES cell through
electroporation and established several ES clones containing
insertional mutations. Using these clones, we generated chimeric mice
and subsequent heterozygous mutants. Of the latter, the Ayu-San112
line, which showed apparent growth retardation after birth, was chosen
for further analysis.11 Southern blot analysis with a
vector fragment and an En-2 fragment as probes indicated that a single
copy of the vector was integrated (Fig 1B). Analysis of the mutated
genomic region and 5'RACE showed that the trapping vector was inserted
into an intron of the CBP gene located between exons containing
nucleotides 3064-3253 and 3254-3373 (Fig 1A). Restriction mapping and
Southern blot analyses showed that gross deletion or rearrangement had
not occurred at the inserted region (Fig 1B). The production of a
truncated protein (residues 1-1084) was predicted from the integration
pattern of the trapping vector into the CBP gene (Fig 1A). This
truncated protein contains the CREB binding domain (residues 462-661),
but not the histone acetyl transferase (HAT) domain (Fig 2C). We
confirmed the expression of a fusion message of CBP and the reporter
gene of pU-San in CBP+/mut and CBPmut/mut mouse
embryos by RT-PCR analysis (Fig 2A).
Furthermore, Western blot analysis using a specific antibody (CBP-A22)
against the N-terminal region of CBP detected a 121-kD protein, which
corresponds to the predicted size of the truncated protein in
CBP+/mut brain and CBPmut/mut embryo, but not
CBP+/+ embryos (Fig 2C). In CBPmut/mut embryos,
wild-type CBP protein was not observed, indicating that only the
truncated protein was produced (Fig 2C). In addition, with a C-terminal
specific antibody (CBP-C20), the smaller fragment was not detected in
three kinds of genotypes (data not shown). The heterozygotes showed
various phenotypes of human Rubinstein-Taybi syndrome and were
fertile,11 but no homozygotes were found among 98 newborn
animals from heterozygous crosses, indicating that the homozygous
mutation induces embryonic lethality. To determine the stage at which
the CBP mutation was lethal, embryos from F3 or F4 intercrosses were
examined. As shown in Fig 2D and E, E9.5 CBP homozygous mutants were
smaller than their wild-type littermates and exhibited an open neural
tube, both of which are seen in p300-deficient mice. Viable CBP
homozygous mutants were not observed at E10.5.
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| Fig 1.
Insertional mutation of the CBP gene. (A) The structures
of the trapping vector (top), the wild-type allele (middle), and
the mutant allele (bottom) are shown. The trapping vector,
pU-San, contains two loxP sequences, a splice
acceptor region (SA) of the mouse En-2 gene, an internal ribosomal
entry site (IRES), a -galactosidase/neomycin phosphotransferase
fusion gene, the SV40 polyadenylation sequence (-geo-pA), and the
pUC19 vector as indicated. The gray boxes represent exons. Restriction
enzyme sites (B, BamHI; E, EcoRI;
H,HindIII; S, SphI), the location of probes (bars)
used to confirm single integration of trapping vector, and the expected
fragment sizes are indicated. Probe A is an
SpeI-BgIII fragment in the SA of the
trapping vector; probe B is an ScaI-XbaI fragment
in pUC sequences of the trapping vector; probe C is the
HindIII-XhoI fragment immediately upstream of
the vector integrated region. The open and closed arrowheads indicate
the location of primers used in RT-PCR for genotyping. (B) Southern
blot analysis of an Ayu-San112 ES clone and a normal TT2 ES cell.
(Left) A blot using HindIII (H) or SphI-digested
(S) DNA hybridized with probe A. (Middle) A blot BamHI- (B)
or EcoRI-digested (E) DNA with probe B. (Right) A blot of
using EcoRI-restricted DNA hybridized with probe C. Molecular-weight makers are shown on the right. The bars indicate the
positions of size marker: 23130, 9416, 6557, 4361, and 2322 bp from the
top.
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| Fig 2.
Characterization of CBP homozygous mutant embryos. (A)
RT-PCR analysis of RNAs isolated from yolk sacs of wild-type embryos,
heterozygous embryos, and homozygous embryos. A
CBP 5'-end forward primer and a 3'-end reverse primer, indicated in
Fig 1A, are used to detect wild-type CBP transcripts. The same CBP 5'
forward primer and a reverse primer in the splice acceptor region are
used to detect CBP/-geo fusion transcripts. (B) Western blotting of
extracts (30 µg protein) from the brains of adult mice (right) and
extracts (5 µg protein) from E9.5 embryos (left) with N-terminal
specific anti-CBP antibodies (CBP-A22). An arrowhead indicates the
265-kD wild-type CBP protein. An arrow indicates the truncated form of
CBP at 121 kD. (C) The schematic structure of putative truncated and
wild-type CBP and p300. Percentages refer to amino acid (aa) identify
between proteins. (D) Phenotypes of wild-type and CBP homozygous mutant
embryos at E9.5. A mutant is smaller and much paler than a wild-type
littermate. The morphology of the cranial region (arrow) of mutants is
distinct from that of wild-type littermates. (E) A CBP homozygous
mutant displays an open neural tube (arrowhead) in the cranial
region.
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The yolk sac of E9.5 CBP homozygous mutant embryos was obviously pale
compared with that of wild-type littermates and the vessels appeared
scarce (Fig 3A). The mutants
themselves were pale and showed arrested growth at E8.75-9.0, allowing
unequivocal identification of homozygous mutants (Fig 2D). Histological
examination of the yolk sac showed that erythroid cells were remarkably
reduced in mutants (Fig 3B). Although the total number of blood cells from the yolk sac was reduced to 20% of control in CBP homozygous mutants (Fig 3C),  and hemoglobin staining showed that the blood
cells in the yolk sac contained a mature stage of primitive erythroid
cells (Fig 3D). Despite normal maturation of red blood cells, CBP
homozygous mutant embryos showed marked anemia and failed to survive
beyond the stage of primitive hematopoiesis. To determine how
development of primitive hematopoiesis is disturbed, we examined a
colony-forming capacity of E9.5 homozygous yolk sac
cells.26 They showed 22% and 69%, respectively, of
erythroid and granulocyte-macrophage (GM) colony-forming capacity
compared with both heterozygous and wild-type counterparts (Table
1).
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| Fig 3.
Phenotypes of CBP homozygous mutant embryos. (A) The E9.5
yolk sac from wild-type (left) and CBP (right) homozygous mutant
embryos. (B) Section of the yolk sac from a wild-type (top) and a CBP
homozygous mutant (bottom) at E9.5. Note the small number of
erythrocytes in the yolk sac derived from CBP homozygous mutants. Scale
bar indicates 100 µm. (C) Numbers of nucleated erythroid cells
present in yolk sac cells from embryos at E9.5. (D) Staining of blood
cells from a wild-type (left) and CBP homozygous mutant (right) yolk
sac with antibodies to  1-globin (top) and -globin (bottom). Scale
bar indicates 30 µm.
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The pattern of the vascular system of CBP homozygous mutants was
studied by staining E9.5 embryos in whole mount or tissue sections with
the anti-PECAM monoclonal antibody, which detects differentiated
endothelial cells.27 We found that all homozygous mutant
embryos failed to form an organized vascular network at E9.5 (Fig 4A
and B). Gross defects of vascular branching
were observed especially in the head and trunk including the P-Sp
region (Fig 4C through F). Because embryos have already finished
"turning" at this stage, abnormalities in the vascular system
cannot be caused by growth retardation of homozygous mutants.
Immunohistochemical analysis showed scarce and disoriented vessels and
a decrease in PECAM-1+ endothelial cells in the brain and
P-Sp regions (Fig 5A through D [see page 2774]).
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| Fig 4.
Vascular network formation in CBP homozygous mutant
embryos. (A and B) E9.5 whole embryos stained with anti-PECAM-1
antibody to visualize all vessel endothelial cells. Note lack of
vascular network formation in CBP homozygous mutant embryos. (C and D)
Higher magnification of the head region of E9.5 embryo. Note lack of
large vessels and smaller vascular branches in the homozygous embryo.
(E and F) Higher magnification of the P-Sp region of E9.5 embryos. Note
poor vascular network formation of P-Sp region in CBP homozygous mutant
embryos.
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| Fig 5.
(A through D) Histological analysis of E9.5 wild-type (A
and C) and CBP homozygous (B and D) mutant embryos stained with
anti-PECAM antibody and counter-stained with hematoxylin. Open neural
tube (arrow in B), disoriented posterior branch of primary head vein
(arrowheads in B), and decreased numbers of blood cells in dorsal aorta
(da; stars in A and B) were observed in the head region of CBP
homozygous mutant embryos. In the trunk, lack of sprouting vessels from
the dorsal aorta or umbilical vein (uv) in parietal mesoderm
(arrowheads in C) and a decrease in PECAM-1+
endothelial cells in the omphalomesenteric artery (oa) and dorsal aorta
(arrows in D) were observed in CBP homozygous mutant embryos. Note
ectopic blood cells in hindgut diverticulum (hd) and coelomic cavity
(cc) in CBP homozygous mutant embryos (stars in D). Scale bar indicates
50 µm.
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To determine how vascular networks are defective in CBP homozygous
mutant embryos, organ culture of P-Sp from E9.5 embryos was undertaken
on OP9 stromal cells.18,22,28 No vascular bed or network formation was detected in P-Sp from CBP homozygous mutant
embryos, while cultures from wild-type embryos developed normally (Fig
6). Because P-Sp from E8.5 wild-type
embryos develops vasculature (data not shown), we concluded that
vascular formation by CBP homozygous mutant embryos is impaired rather
than delayed. Transcripts for VEGF165 and VEGF receptors,
Flk-1 and Flt-1, remained unchanged in embryos homozygous for the CBP
mutation (data not shown). Addition of 100 ng/mL VEGF to this culture
slightly rescued vascular bed and network formation in mutants (Fig 6).
However, this recovery was less than that seen in wild-type littermate explants cultured with or without VEGF.
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| Fig 6.
Vasculo-angiogenesis in P-Sp culture. P-Sp explants
derived from E9.5 CBP homozygous mutant embryos and CBP wild-type
littermates were cultured on OP9 stromal cells. Note that vascular bed
and vascular network formation were defective in mutant embryo explants
compared with that of wild-type littermates. VEGF (100 ng/mL) was added
to the culture system as noted above. Vascular bed (vb) and vascular
network (vn) formation were partially rescued in CBP homozygous mutants
by addition of VEGF. However, note that vascular bed and network
formation of P-Sp explants from CBP homozygous mutant embryos was
poorer than that of wild-type littermates. Scale bar indicates 50 µm.
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To examine definitive hematopoiesis of the mutant embryos, we performed
coculture of E9.5 P-Sp on OP9 cells. After 7 days of culture, the
colony-forming cells (CFCs) of nonadherent cells was examined in a
methylcellulose medium. As shown in Table
2, the total number of CFCs in cultured
P-Sp cells from homozygous mutants was markedly reduced compared with
those from wild-type P-Sp. Addition of 100 ng/mL VEGF to this
culture partially rescued erythroid colony formation in mutants
(Table 2), as the vascular formation was recovered. However, the number
of CFCs did not reach the level of wild type.
| |
DISCUSSION |
In this report, we show that the transcription cofactor, CBP, is
essential for embryogenic hematopoiesis and vasculo-angiogenesis. Because the development of hematopoiesis is associated with that of
vasculo-angiogenesis, the phenotypes of CBP mutants are noteworthy.
Hematopoiesis consists of primitive hematopoiesis in the yolk sac and
definitive hematopoiesis which develops in the P-Sp region.15-18 Here, we show a reduction in hematopoietic
progenitor cells in the yolk sac and P-Sp of CBP homozygous mutant
mice. Therefore, anemia seen in mutant mice is due not to a loss of blood but to an intrinsic defect in proliferation of progenitor cells
in both primitive and definitive hematopoietic system. In CBP mutant
mice, the erythroid lineage was more susceptible than the GM lineage
(Tables 1 and 2). It has been shown that the E1A binding region of CBP
binds to the zinc finger region of GATA-1 and that CBP markedly
stimulates GATA-1's transcriptional activity.29 It is also
likely that a defect in vascular endothelial cells may cause the defect
in hematopoiesis, because these cells play a critical role in the
hematopoietic microenvironment. In vitro definitive hematopoiesis of
homozygous embryos was partially rescued by addition of VEGF (Table 2).
To determine whether the hematopoietic defect is intrinsic or a
consequence of defective angiogenesis, mouse chimera analysis will be required.
Gross defects in vascular branching is evident in CBP homozygous mutant
mice at E9.5. The number of PECAM-1+ endothelial cells is
significantly decreased. These abnormalities are confirmed by in vitro
coculture of P-Sp on OP9 stromal layers. Embryonic vessel formation
consists of two steps: vasculogenesis, the de novo organization of
endothelial cells into vessels, and angiogenesis, the continued
expansion of the vascular tree as a result of endothelial cell
sprouting from existing vessels. In the P-Sp explant culture system,
both vasculogenesis and angiogenesis can be observed in
vitro.22 Both processes were defective in P-Sp cultures
from CBP mutant mice. Addition of VEGF to mutant cultures partially
rescued these defects. Expression of angiogenic factors such as VEGF
and angiopoietins and their receptors, Flk-1, Flt-1, and TIE2, in
mutant mice were not changed compared with wild type (data not shown).
Therefore, we conclude that the defect in vascular network formation in
CBP homozygous mutant embryos is due to the inability of putative
angioblasts in P-Sp to proliferate and differentiate. Recently, it has
been reported that CBP transactivates by interacting with Ets-1, which
is expressed in migrating and sprouting endothelial
cells.30 Moreover, Smad proteins, which are involved in
mediating the transforming growth factor- (TGF-)-response can
functionally interact with CBP in endothelial cells.31 It remains to be clarified whether homozygous mutants exhibit altered expression of Ets-1 and/or Smads during embryonic angiogenesis.
In our study, CBP homozygous mutants, which made a truncated CBP
protein (residues 1-1084) showed embryonic lethality. Investigation of
the phenotypes of CBP homozygous null mutants compared with our
disrupted CBP mutants will show the function of the N-terminal or
C-terminal domain of CBP in vivo. Although specific details of the
phenotype of CBP null mutants have not yet been published, the mice die
at E8-10 and display neural tube defects, similar to our CBP disrupted
mutants.13
In summary, mice homozygous for a truncated form of CBP mutants die
between E9.5 and E10.5 and exhibit impaired hematopoiesis and
vasculo-angiogenesis. The P-Sp culture shows an autonomous abnormality
of putative endothelial precursors, which may induce the
microenvironmental defect in hematopoiesis. Our analyses show that CBP
plays an essential role in hematopoiesis and vasculo-angiogenesis in
mammalian cells.
| |
ACKNOWLEDGMENT |
We thank Y. Kiyonaga, M. Tokushima, and I. Kawasaki for technical assistance.
| |
FOOTNOTES |
Submitted October 20, 1998; accepted February 1, 1999.
Y.O. and N.T. made an equal contribution to this paper.
Supported by grants from the Ministry of Education, Science and Culture
of Japan, a grant from the Yamanouchi Foundation for Research on
Metabolic Disorders, a grant from the Osaka Foundation for Promotion of
Clinical Immunology, and a grant from the Science and Technology Agency.
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 reprint requests to Toshio Suda, MD, Department of Cell
Differentiation, Institute of Molecular Embryology and Genetics,
Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860-0811, Japan; e-mail: sudato{at}gpo.kumamoto-u.ac.jp.
| |
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ABSTRACT
INTRODUCTION