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The Role of BMP-4 and GATA-2 in the Induction and Differentiation of
Hematopoietic Mesoderm in Xenopus Laevis
By Mitsugu Maeno, Paul E. Mead, Clair Kelley, Ren-he Xu, Hsiang-fu Kung, Atsushi Suzuki, Naoto Ueno,
and Leonard I. Zon
Vertebrate embryonic blood formation is regulated by factors that participate in dorsal-ventral patterning and mesoderm induction. The GATA-binding transcription factors are
required for normal hematopoiesis and are expressed during
gastrulation when ventral mesoderm (VM) is induced to
form blood. Based on the recent demonstration that bone
morphogenetic protein (BMP-4) is a potent ventralizing factor and inducer of hematopoietic tissue, we hypothesized
that GATA-2 could be induced or activated by BMP-4. Here
we demonstrate that BMP-4 can stimulate GATA-2 expres-
sion, and that expressionofa
dominant negative BMP-4
receptor can suppressGATA-2 induction by BMP-4 in ventral
mesoderm. Over-expressionof GATA-2 in ventral mesoderm
leads to increased globin production and forced expression
of GATA-2 in primitive ectoderm adjacent to ventral mesoderm also stimulates globin expression. Our results suggest
that BMP-4 and GATA-2 can function in two adjacent germ
layers, mesoderm and ectoderm, to participate in blood cell
formation during embryogenesis.
0 1996 by The American Society of Hematology.
V
cells can stimulate hematopoietic differentiation. VMZ explants from stage 10 express globin RNA and protein after
48 hours of culture.‘ When VM is isolated from the VMZ
and cultured for 48 hours, no globin protein is expressed
(although globin mRNA is d e t e ~ t e d ) . ’ ~ During
. ~ ~ . ~ ’gastrulation, animal pole ectoderm comes into apposition with VM
(Fig IA). CO-culture of stage 10 (early gastrula) animal pole
tissue with the VM leads to increased globin protein expression with VM (Fig IB); however, stage 7 (early blastula)
animal pole tissue cultured with VM leads to little, if any,
globin protein (Fig 1C). This suggests that factors in the
stage 10 animal pole regulate blood formation. When stage
7 animal pole explants derived from embryos injected with
BMP-4 RNA are co-cultured with VM, increased globin
protein is detected (Fig lD).14CO-cultures of stage 10 animal
poles with VM that expresses the dominant negative BMP4 receptor demonstrate no globin protein synthesis, These
data suggest that BMP-4 receptor signalling is required for
stimulation of hematopoietic differentiation by the animal
cap.
BMP-4 appears to directly induce VM and also supports
hematopoietic differentiation via the ectoderm of the gastrula
ERTEBRATE BLOOD CELL development involves
the induction of ventral mesoderm and the subsequent
proliferation and differentiation of hematopoietic progenitors.’ In amphibians, mesoderm in the ventral marginal zone
(VMZ) is fated to form the blood
and the induction
of this mesoderm correlates withthe initial expression of
DNA-binding proteins that regulate hematopoietic-specific
gene transcription such as GATA-1 and GATA-2? Disruption of the GATA-l or GATA-2 locus in mice has demonstrated that both factors are required for normal hematopoieis.^,' GATA-1 is localized to the ventral region of the
Xenopus embryo and precedes the expression of embryonic
globins, and GATA-2 is expressed in the ventral-lateral ectoderm and mesoderm, blood cells, and the central nervous
system.’.8 GATA-2 is expressed at a low level as a maternal
RNA and zygotic transcription is evident at the onset of
gastrulation.’.’ GATA- 1 levels increase and GATA-2 levels
decrease as hematopoietic cells differentiate within the ventral blood island. VMZ explants express both GATA-I and
GATA-2 RNA in a similar spatial and temporal pattern of
expression to that of the whole embryo.* Culture of animal
pole ectoderm surprisingly demonstrates an autonomous expression of GATA-1 and GATA-2, and this expression is
not maintained by culture alone or with added fibroblast
growth factor jbFGF) or a~tivin.’.~
Thus, other factors are
likely to induce hematopoietic mesodermand
regulate
GATA-binding protein expression.
One candidate inducer for ventral mesoderm (VM) is bone
morphogenetic protein (BMP-4).9”6 BMP-4 is expressed in
animal pole cells during blastula stages and localizes to the
ventral and lateral marginal zone during gashulation.”,’’.’4,’5
Forced BMP-4 expression in early Xenopus embryos leads
to hyperventralization (lack of dorsal structures) and induces
globin RNA expression in animal pole explants.9-”Xenopus
BMP-4 receptors have been recently isolated and expression
of a dominant negative BMP-4 receptor leads to decreased
blood formation.”.””5 Through gene targeting experiments
in mice, both BMP-4 and its receptor have been shown to be
required for ventral mesoderm formation.”.” Furthermore,
BMP-4 has been shown to induce globin expression in murine embryonic stem cell cultures.” Thus, BMP-4 or related
family members are likely to have a role in the induction of
hematopoietic mesoderm.
Recent studies have also demonstrated that animal pole
Blood, Vol 88, No 6 (September 151, 1996: pp 1965-1972
Fromthe Department of Biology, Faculty of Science, Niigata
Universiq, Niigata, Japan; the Division of Hematology, Department
of Pediatrics, Howard Hughes Medical Institute, Children’s Hospital, Boston, MA; the Laboratovy of Biochemical Physiology, Department of Basic Sciences, National Cancer Institute-Frederick Cancer
Research and Development Center, Frederick, MD; and the Faculty
of Pharmaceutical Science, Hokkaido University, Sapporo, Japan.
Submitted January 11, 1996; accepted April 29, 1996.
Supported by National Institutes of Health Grant No. ROIHLA8801. Zon is an Assistant Investigator and Mead is an Associate
of the Howard Hughes Medical Institute.
Address reprint requests to Leonard I. Zon, MD, Division of HentatoIogy, Department of Pediatrics, Howard Hughes Medical Institute, Children’s Hospital, 300 Longwood Ave, Enders 780, Boston,
MA 02115.
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 I 8 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-497I#6/8806-0019$3.00/0
1965
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1966
MAENO ET AL
A
v@D
NormalGastrula
Embryo
B
.c
st. 10
GlobinProteinExpression
C
st. 7
si. 10
Expression
Protein
GlobinNo
RNA (i.e. BMP-4or
GATA-2)
D
b
Vegetal
One
or
Two
Cell
Embryo
St.
7
1
GlobinProteinExpression
stage embryo. GATA-2 is expressed in both the VM and
ectoderm during gastrula stages and may direct hematopoiesis in these two germ layers, similar to BMP-4. Here, we
demonstrate that BMP-4 induces increased levels of GATA2 and the dominant negative BMP-4 receptor suppresses
GATA-2 expression in the VM. We also demonstrate that
forced expression of GATA-2 in the animal pole stimulates
VM to form globin. Taken together, these results suggest
that GATA-2 not only functions in a cell autonomous manner during the induction of the blood program, but may also
have a role in the animal pole to regulate the differentiation
of blood.
MATERIALS AND METHODS
Immunohistochemistry and in situ hybridization methods. Whole
embryo immunohistochemistry was performed as described.' Embryos previously fixed in 3.7% formaldehyde and stored in methanol
were gradually rehydrated in phosphate buffered saline (PBS). These
were then incubated in a 115 dilution of monoclonal antibody, U-27,
supernatant (provided by C. Katagiri, Hokkaido University, Sapporo,
Japan). This antibody recognizes larval globins. Peroxidase conju-
Fig 1.
experiments
co-culture
Schematic
using
of
assay. (A) During gastrulation, AP ectoderm comes
into contactwith VM. This AP ectoderm may stimulatehematopoieticdifferentiation.
(B) When APs
stage fromco-cultured
10 are
with
stage
V M from
10,
increased hematopoietic differentiation is evident in
the VM, as shown by globin production.(C) When a
stage 7 cap is combined with VM, no globin expression is detected.(D)Assay for factors that stimulate
D hematopoieticdifferentiation. Capsareloaded with
RNA at the one or two cell stages and removed at
s1. 10
stage 7. Uponco-culture,increasedhematopoietic
differentiation is demonstratedbyglobinproduction. As demonstrated here,B M P 4 and GATA-2 can
each stimulate globin expression in this assay.
0
gated secondary antibodies (Jackson labs No. 115036062) were used
at a 1:250 dilution. Detection was performed with HzOl and DAB
(Polysciences, Wamngton, PA). Whole embryo in situ analysis for
GATA-2 expression was performed as previously described using
antisense, digoxigenin-labeled, full-length GATA-2 RNA as probe.'
Western blot analysis. Western blot analysis for globin expression was performed as previously described,2"."using the monoclonal antibody L5.41. In each experiment, explants from at least
five embryos were pooled, and an equivalent of one embryo was
loaded onto a 3M urea-18% sodiumdodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE). Detection used the ECL system
(Amersham).
RNA injections. Embryos were injected atthe two or four cell
stage with the vectors: BMP-4/pSP64T, delta TRFll/pSP64T (the
dominant negative BMP-4 receptor), and GATA-2PGEMHE. The
TRFll receptor is the mouse ALK-3:' and the dominant negative
construct and its effect on Xenopus development has been described
previo~sly.'~,'~
BMP-4 and BMP-2 can compete for binding to the
TRFll receptor, but activin and TGFPl cannot. The BMP-4and
the delta TRFl1 plasmids were each linearized with EcoRI and used
SP6 polymerase (Ambion). The GATA-2 plasmid was linearized at
XbaI and usedT7 RNA polymerase (Ambion) for RNA transcription.
RT-PCR analysis. RT-PCR analysis for gene expression was
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BMP-4 AND GATA-2 REGULATE BLOOD FORMATION
1967
performed according to standard protocols.',' RNA was isolated from
ten or more animal caps or ventral marginal zone recombinants. A
totalof 0.5 pg RNA was subjected to reverse transcription. One
tenth volume of this cDNA was amplified with primers specific for
BMP-4" and for GATA-2'.' as published, half of this product was
loaded on a 5 8 polyacrylamide gel. To quantify the RNA amount
used for each analysis, EFla expression was examined by Northern
blot analysis. Densitometry was performed with a scanning imager
(Personal Densitometer; Molecular Dynamics Japan, Tokyo, Japan).
For the demonstration of GATA-2 RNA quantification experiment,
RNA
was
synthesized in vitro from the linearized GATA-2/
PGEMHE vector and an optical density measurement was obtained
to quantitate the amount of synthesized RNA. The RNA was serially
diluted and RT-PCR analysis was performed as described above.
Cell cultrtre. Animal cap explants or recombinants of animal
pole tissue with ventral mesoderm were prepared as previously described.""" The restricted area of prospective ventral mesoderm (VM
in Fig 1) was prepared by removing all the cells above the blastocoel
floor level.'" The explants excised were cultured in Steinberg's solution for the designated periods.
Each experiment was repeated multiple times to ensure reproducibility of results. The repeated experiments involved deriving new
embryonic explants and subjecting these tissues to either RT-PCR
or Western blot analyses. In all cases, similar results were obtained
compared withthosethat
are presented in each figure.We have
stated the number of repeat experiments performed in each figure
legend.
~
Ventral-lateral -view
~
Ventral view
v
RESULTS
BMP-4 expression leads to extensive globin expression in
the embryo. BMP-4 induces ventralization of the whole
embryo and has been shown to activate globin RNA expression in the animal pole, which normally gives rise to ectod e ~ m . ~To
' ' determine
~
the effect of BMP-4 on globin protein
expression in the whole embryo, BMP-4 RNA was injected
at the one cell stage and embryos were allowed to reach
stage 30' Whole embryo immunohistochemistry with a
monoclonal antibodyto embryonic globin demonstrated that
embryos injectedwithBMP-4 exhibited globin expression
throughout the embryo except in the most dorsal dome (Fig
2, the arrows delineate the dorsal border of the blood island).
This is in contrast to a typical V-shaped blood island seen
in embryos injected with a control RNA (&galactosidase).
This data suggests that BMP-4 expression leads to the cornmitment of more mesoderm of the embryo to form hematopoietic tissue.
Sparial regulation of GATA-2. The equatorial or marginal zone (MZ) of the gastrula gives rise to the mesodermal
tissues. GATA-2 is first expressed in the ectodermal animal
pole (AP) and subsequently in both the ventral and dorsal
marginal zones of embryos during gastrulation.'.' Because
these MZ explants included an outer cell layer that could be
contaminated with animal pole cells, we studied GATA-2
expression in VM and dorsal mesoderm (DM) that has been
completely separated from these outer cell layers. A sensitive
semi-quantitative RT-PCR analysis that can detect levels of
GATA-2 RNA as low as 0.8 pg was usedR(Fig 3A). The level
of GATA-2 RNA expression detected in whole embryos by
this assay correlates with the expression detected by whole
embryos in situ analysis.'.' For instance, the level of GATA-
Fig 2.
~ ~ p expression
- 4
leads to substantialglobinexpression
in the Xenopus embryo. Wholeembryoimmunohistochemistry for
globinexpression in embryos injected with control p-galactosidase
RNA (upper in each panell and BMP-4 RNA (lower in each panel). The
top panel is a ventral-lateral view and the bottom panel is a ventral
view. BMP-4 expressionleads to globin expression throughout the
embryo exceptin the most dorsal dome (the arrows delineate the
dorsalboundary of the blood island).
2 RNA increases substantially during neurula stages and then
decreases during further development (Fig 3B). In the early
gastrula embryo, GATA-2 is expressed in the AP and VM,
and at a low level in the DM (Fig 3C).
To understand the regulation of GATA-2 in the AP cells,
GATA-2 expression was examined by RT-PCR in isolated
AP ectoderm at various stages of development. AP from the
early blastula (stage 7) express very low levels of GATA-2
(Fig 3D) in contrast to the level expressed in a stage 10
animal pole explant. The removal of the AP at stage 7 may
prevent the cells from being stimulated by factors from the
rest of the embryo that regulate GATA-2 expression. To
examine this, stage 7 AP explants were explanted and cultured until stage IO. GATA-2 in the cultured AP was expressed at a low level, comparable with that of stage 7 AP.
This suggests that GATA-2 expression is induced by other
regions of the embryo during the blastula and gastrula stages.
Relationship between BMP-4 and GATA-2. Based on the
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MAENO ET
1968
XGATA-2
A
B
0
7
c;
f
v)
+;
v)
XGATA-2
EF1 a
-
XGATA-2
--t
--c
C
D
putative role of BMP-4 as a VM inducer, we studied whether
BMP-4 could induce GATA-2 expression. Embryos were
injected at the one cell stage with BMP-4 RNA and grown
until tailbud stage 28. In situ analysis of these ventralized
embryos showed GATA-2 expression throughout the mesodermal and ectodermal layers, except where small remnants
of dorsal tissue are evident (Fig 4A). Embryos ventralized
by UV-irradiation also have expanded GATA-2 expression,
demonstrating that GATA-2 expression reflects the ventral
character of the embryo.2
Fig 3. GATA-2 expression in
regionsoftheembryo
as detected by RT-PCR analysis. (A)
GATA-2 RNAwas synthesized in
vitro and the indicated dilution
was subjected t o RT-PCR analysis. The assay can detect 0.8 pg
GATA-2 RNA.(B)RNA from each
stage of development was subjected t o RT-PCR analysis. (C)
GATA-2 is expressed in the presumptive VM region and AP tissue, but not in the presumptive
DM region at stage lo'/,. Similar
results were obtainedin two independent experiments. (D)
GATA-2RNA expression in AP
cells. AP cells explanted at stage
7 barely express GATA-P, while
explanted stage 10 AP cells express abundant GATA-2 RNA.
Similar results were obtainedin
four independent experiments.
When AP cells are explanted at
stage 7 and cultured t o stage 10,
little GATA-2 expression is detected. Similar results were obtained twice.
To define the regulation of GATA-2 expression, AP regions derived from embryos injected with BMP-4 RNA were
removed at stage 7 and cultured until early stage 10. BMP4 leads to a marked increase in GATA-2 RNA level compared with the level detected in A p from uninjected embryos
or from embryos injected with RNA encoding BMP I B (Fig
4B).24Thus, BMP-4 can induce GATA-2 expression in animal pole ectoderm.
To examine a potential feedback mechanism, the ability
of GATA-2 to induce BMP-4 was evaluated in AP explants.
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GATA-2
BMP-4 AND
BLOOD FORMATION
1969
BMP-4 was detected in untreated AP explants, as previously
described,~1.!2.~4.~~
while injection of GATA-2 RNA did not
lead to increased BMP-4 RNA levels in the AP. Thus,
GATA-2 is activated downstream of BMP-4 signaling during
normal development.
Effects of thedominant negative BMP-4 receptor on
GATA-2 expression. To determine whether BMP-4 signaling is required for the induction of GATA-2, the effect of a
dominant negative BMP-4 receptor was examined using VM
or AP explants. The dominant negative receptor (delta
TRFI 1) is derived from the mouse ALK-3,” and the dominant negative construct and its effect on Xenopus development has been described previou~ly.’~.’~
BMP-4 and BMP2 can compete for binding to the TRFl1 receptor, but activin
and TGFPI cannot. Expression of the dominant negative
BMP-4 receptor in VM significantly decreased GATA-2 expression by 21-fold (based on densitometry). Thus, BMP-4
signaling is required to stimulate GATA-2 expression and
hematopoietic mesoderm formation (Fig 5). In contrast, the
dominant negative BMP-4 receptor slightly suppressed endogenous GATA-2 RNA expression in AP explants by approximately three-fold. These findings suggest that regulation of GATA-2 expression is distinctly different in the
animal pole compared with that in the VM.
Role of GATA-2 during early development. To determine whether forced GATA-2 expression could stimulate
hematopoiesis in Xenopus, GATA-2 RNA was injected into
both cells of two cell embryos, VM was explanted at stage
IO, and globin expression was determined at 48 hours of
culture by Western blot analysis using a specific embryonic
globin monoclonal antibody.” As shown in Fig 6 , GATA-2
is able to stimulate low levels of globin expression in VM.
Previous studies have demonstrated a stimulation of hematopoietic differentiation in the VM by AP ectoderm20,2’(Fig
1 ). This AP ectoderm expresses GATA-2 and becomes juxtaposed to the VM during gastrulation, adjacent to mesoderm
that will give rise to blood.’ In addition to the role of GATA-
2 in hematopoietic progenitors, GATA-2 may also regulate
an ectodermal program that promotes globin expression. To
test this hypothesis, GATA-2 RNA was injected into embryos at the two cell stage, APs were explanted at stage 7,
and co-cultured with stage IO VM. While uninjected stage 7
AP explants do not promote globin expression, surprisingly,
stage 7 AP from embryos injected with very low levels of
GATA-2 RNA (8 pp) stimulated globin protein synthesis.
The level of globin detected was comparable with that induced by stage I O animal caps (Fig 1 and data not shown)
and was much higher than in VM explants from embryos
injected with GATA-2 RNA. These studies suggest a role for
GATA-2 in adjacent ectoderm for the production of globin.
1
+
Fig 4. BMP-4 regulates GATA-2 expression. (A) Whole embryoin
situ analysis for GATA-2 expression in an embryo loaded with BMP4 RNA. The embryo is allowedt o reach control stage 28 (see insert;
the intense purple color indicates ventral
expression of GATA-2). Embryos injectedwith BMP-4 RNA lack dorsal structures. The staining of
GATA-2 is ina radially symmetric pattern. Some partially
ventralized
embryos have GATA-2 expression in an expanded pattern reminiscent of the control embryo(in the insert). (B) BMP-4 induces GATA2 expression in the early gastrula animal pole. Animal caps were
loaded with BMP-4, GATA-2 RNA, or the controlBMP-l B RNA. Animal
caps were dissected at stage 7 and allowed t o develop until stage
lo’/., at which timeGATA-2 expression and BMP-4 expression were
determined by RT-PCR analysis. E F l a expression from thesame RNA
samples was determined by Northern blot
analysis. Note thatGATA2 was substantially induced by B M P 4 RNA injection, but GATA-2
RNA expression cannot increase the expression of BMP-4. Relative
ratios of GATA-2 expression t o E F l a expression based on densitometry is 0.109, 0.165, 1.715, 1.673 for each of the lanes respectively in
the figure. Relative ratios of BMP-4 expression to EFla expression
based on densitometryare 0.470,0.596,3.180, and 0.235 for each of
the lanes, respectively, in the figure. Similar results were obtained
in four independent experiments.
c
-
-v-
XGATA-P
XBMP4
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MAENO ET AL
1970
a
n
d
7
.-0
G
c
t
Z
n
0
7
C
",
0
z
0)
C
v)
.0
z
0
z
-XGATA-2
itive and definitive hematopoietic progenitors.' In vitro cultures of homozygous GATA-2- ES cell lines demonstrate a
substantial decrease in progenitor number and these homozygous lines fail to contribute to hematopoiesis in vivo in
chimeric mice.7 In support of this important role of GATA2 in embryonic blood formation, we have demonstrated that
GATA-2 is expressed in the early VM thatwillbecome
blood.* Furthermore, forced expression of GATA-2 in the
VM leads to a slight increase of globin protein expression
after culture for 48 hours. Thus, as shown in the mouse,
GATA-2 acts in a cell-autonomous manner during normal
blood formation.
In addition to expression in the VM, GATA-2 is abundantly expressed in the ventral ectoderm.*.' This ectodermal
expression pattern is also
exhibited in the zebrafish, in which
the ventral yolk syncytial layer expresses high levels of
GATA-2 RNA.'6 Early hematopoietic progenitors contact
this layer, similar to the contact of ventral hematopoietic
mesoderm and ventral ectoderm in Xenopus. Previous studies demonstrate that the ventral ectoderm stimulates hematopoietic differentiation.2" Our studies, using AP and VM
co-cultures, showed that GATA-2 expression in the AP ectoderm increased globin expression within the VM. Thus, in
addition to a hematopoietic cell autonomous role, GATA-2
Fig 5. Dominant negative BMP-4 receptor expression leads t o a
decrease of the endogenous GATA-P expression in VM. Dominant
negative BMP-4 receptor RNA was injected either
into AP or VM, and
explants of animalcaps, ventral marginal tissues, or whole embryo
was cultured fromstage 10'1, t o stage 14, at which theendogenous
GATA-2 expression would be maximum. GATA-P expression in the
explants was determined RT-PCR.
by
EFla expression from thesame
RNA samples were determined by Northern blot analysis. Relative
ratios ofGATA-2 expression to EFla
expression based on densitometry are 3.163, 0.996, 1.657, 0.078, 0.600, 0.562
for each of the lanes,
respectively, in the figure. These AP and VM experiments wereperformed three timesand twice, respectively, with similar results.
DISCUSSION
BMP-4 and hematopoietic mesoderm. The induction of
blood formation involves both the commitment of mesoderm
to form hematopoietic tissue and the subsequent differentiation of hematopoietic lineages. VM is induced during or
before gastrulation' and BMP-4 appears to have a prominent
role in this process?"' BMP-4 is first expressed in the AP,
and then localized to the ventral and lateral MZ during gastrulation. Forced expression of BMP-4 ventralizes whole embryos and BMP-4 can rescue partially dorsalized embryos
caused by lithium chloride treatment." Furthermore, BMP4 has been shown to induce globin RNA synthesis in the
AP. Basedon our studies of VM and AP recombinants,
BMP-4 expressed in the AP can promote globin production
of adjacent hematopoietic
In higher vertebrates,
BMP-4 can stimulate globin expression in ES cell culture^.'^
BMP-4 and its receptor have also been shown to be required
for normal VM formation.I7.l8BMP-4 thus appears to participate in both the induction and differentiation of VM.
The role of GATA-2. Disruption of GATA-2 in mice
leads to a substantial proliferative defect in developing'prim-
Globin
+
Fig 6. GATA-2 induces increased globin production. GATA-2 RNA
(0.2 ng) was injected into APs at the two cell stage. Embryos were
allowed t o develop t o stage 7, at which timeAP were removedand
co-cultured with VM fromstage 10 embryo. After 2 days of culture,
explants were harvested t o detect globin protein by Western blot
analysis. GATA-2 is a potent inducer of globin production (VM+ st.7
AP XGATA-2). Combination explantof VM withstage 10 AP tissue
(VM st.10 AP) leads t o high levels of globin production. GATA-2
XGATA-P) led t o slightly increased levels of
RNA injection (VM
globin proteincompared with VM
alone. Co-culture of GATA-2 loaded
VM with stage 7 AP (VM XGATA-2 st.7 AP) failed to stimulate
globin expression. In contrast, co-culture of GATA-2 loaded caps with
the VM (VM st.7 AP XGATA-2) led t o potent stimulationof globin
production. Lanes S1, S2, and S3 contain the lysates of 500,2,500,
and 12,500 erythrocytes from stage 54 larvae. Similar results were
obtained in three independent experiments.
-+
-
+
+
-
+
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BMP-4 AND GATA-PREGULATE
BLOOD FORMATION
1971
and the “indirect” stimulation of BMP-4 on hematopoietic
may also function in this ectodermal (or stromal) layer in
differentiation by the AP cells.
a non-cell autonomousmannertopromotehematopoietic
BMP-4 and activin are members of the TGF-0 family.
differentiation. Because the functional equivalent to this
venTheir receptors are formed as multimeric complexes contral ectoderm is not obvious in higher vertebrates, it is not
sisting of type I and I1 receptor^.^' Recent studies have demeasy to determine whether GATA-2 has a non-cell autonoonstrated that activin and BMP-4 can bind to the same type
mous role in the hematopoietic development of higher speI and possibly type I1 receptors based on in vitro studies (J.
cies.Although difficult todemonstrate, stromal celllines
Massague, personal communication, May 1995). It is thus
from the GATA-2(-/-) embryos may have a decreased efpossible that erythroid differentiation may occur on stimulaficiency of ability to support hematopoiesisor hematopoietic
tion of either BMP-4 or activin receptor subunits.
stem cell development.
During the cell movementsof gastrulation, the ventral AP
Regulation of GATA-2 by BMP-4. BMP-4 issufficient
ectodermandmesodermbecomejuxtaposed,andBMP-4
to induce AP cells to express abundant GATA-2; however,
apparently acts in these twoadjacent layers of tissue to affect
ourstudies with thedominantnegativeBMP-4receptor
hematopoietic induction and differentiation. BMP-4 expresdemonstrate that BMP-4signalingis
notnecessary
for
sion in the MZ stimulates the formation
of VM, heralded by
GATA-2 expression in AP cells. Thus, the mesodermal and
the induction of specific regulators
of hematopoietic gene
ectodermal expression of GATA-2 is regulated by separate
transcription such as the GATA-bindingproteins. By the end
mechanisms. Our AP explant experiments in Fig 3 suggest
of gastrulation, AP tissue expressing BMP-4 and GATA-2
that the ectodermal expression appears to be stimulated by
comes in contact with the VM. BMP-4 and GATA-2 in the
factorssupplied by the embryo between stages 7 and 10.
AP promotes blood island formation and globin expression
These inducing factors are likely to be distinct from BMPin the VM. BMP-4 and GATA-2 may function in both the
4, based on our results that the dominant negative BMP-4
VMZ and AP ectoderm to stimulate blood formation. This
receptor does not suppress endogenous GATA-2 expression
is analogous to sonic hedgehog’s functionin the determinainthe AP. The regulationof GATA-2 in the ectodermal
tion of the adjacent notochord and neural floor plate.36 Our
and mesodermal germ layers may be regulated by different
results are complementary to those of Zhang and Evans3’
signaling cascades or alternative gene regulation, suchthe
as
who demonstrate that zygotic BMP-4 receptor signaling is
activationofdistinct
GATA-2 promoter elements in each
required for normal hematopoiesis in Xenopus. Future studgerm layer.
ies will focus on isolating an AP factorstimulated by GATABMP-4 and GATA-2 each function in adjacent mesoderm
2 that regulates hematopoietic differentiation and to define
and ectoderm to promote hematopoietic differentiation.
other inducers of GATA-2 in the AP ectoderm.
BMP-4 has been shown to have two effects on the
induction
of blood formation. First, it can induce VM formation, and
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Zon
LI:
Developmental
biology of hematopoiesis.Blood
sion in VM. These direct and indirect roles for BMP-4 may
86:2876,
1995
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1996 88: 1965-1972
The role of BMP-4 and GATA-2 in the induction and differentiation of
hematopoietic mesoderm in Xenopus laevis
M Maeno, PE Mead, C Kelley, RH Xu, HF Kung, A Suzuki, N Ueno and LI Zon
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