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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4128-4137
Cooperative Effects of Growth Factors Involved in the Induction of
Hematopoietic Mesoderm
By
Tara L. Huber,
Yi Zhou,
Paul E. Mead, and
Leonard I. Zon
From the Division of Hematology/Oncology and Howard Hughes Medical
Institute, Children's Hospital, Harvard Medical School, Boston, MA.
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ABSTRACT |
Hematopoietic induction occurs on the ventral side of
Xenopus gastrulae and is thought to be triggered by the growth
factor bone morphogenetic protein 4 (BMP-4). To characterize this
process, we developed a quantitative and sensitive assay for the
induction of erythroid cells from totipotent ectoderm of the embryo.
When high doses of BMP-4 were used in this explant assay, few erythroid cells were detected. In contrast, large numbers of differentiated erythroid cells were induced when ectoderm was treated with BMP-4 and
the mesoderm inducers, activin, or fibroblast growth
factor (FGF). Ectopic expression of GATA-1 also induced abundant
erythroid cells in ectoderm treated with bFGF. This induction of
erythroid cells by GATA-1 was blocked by coexpression with a dominant
negative BMP-4 receptor, showing that GATA-1 requires the BMP signaling cascade to function. These results suggest that BMP-4 requires mesoderm
induction to generate a program of gene expression, which regulates the
specification of hematopoietic mesoderm by GATA factors.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIESIS INVOLVES the
proliferation and differentiation of hematopoietic stem
cells (HSC) to yield erythroid, myeloid, and lymphoid
lineages.1 In Xenopus, all HSCs (primitive or
definitive) are derived from the ventral mesoderm of the
gastrula2 and populate two sites of the embryo, the ventral
blood island (VBI) and the dorsal lateral plate (DLP).3-7
These sites are analogous to the yolk sac and AGM (aorta, gonads,
mesonephros) region of higher vertebrates,
respectively.8-11 Although factors involved in the
proliferation and differentiation of hematopoietic lineages have been
found and characterized, the factors that regulate the initial
specification of HSCs during embryogenesis remain elusive. These
factors presumably act downstream of early mesoderm patterning events
in the embryo.
Bone morphogenetic protein 4 (BMP-4) is a transforming growth
factor- (TGF-) family member that patterns ventral mesoderm. Injection of BMP-4 RNA or expression plasmids into embryos results in
an expansion of the ventral and posterior region of the embryo and a
decrease in dorsal and anterior structures.12-15 Ectopic expression of BMP-4 in totipotent ectodermal explants, called animal
caps, induces expression of the hematopoietic-specific transcription
factors GATA-1, GATA-2, and stem cell leukemia
(SCL)16,17 and globin.17 Nevertheless, the dose
of BMP-4 RNA required to produce erythroid cells or globin protein in
these explants is extremely high and may not be
physiologic.17-19 The explants may require other factors to
cooperate with BMP-4 to regulate the early events of blood formation in
vivo.
To test this hypothesis, we evaluated the role of BMP-4 and GATA-1 in
embryonic hematopoiesis using an assay for the induction of erythroid
cells from explants of primitive embryonic ectoderm. These explants
were dispersed and stained with o-dianisidine to visualize
single erythroid cells. This assay is sensitive, reproducible, and
quantitative. BMP-4 overexpression induced low numbers of erythroid
cells; however, in the presence of mesoderm inducers such as fibroblast
growth factor (FGF) or activin, large numbers of erythroid cells were
detected. GATA-1 overexpression induced abundant erythroid cells in the
presence of FGF. Coinjection of GATA-1 and a dominant negative BMP
receptor did not induce blood in animal explants treated with FGF,
showing that GATA-1 requires the BMP signaling cascade to function.
Blood formation during vertebrate embryogenesis may thus require at
least three distinct functions: mesoderm induction (activin or FGF),
mesoderm patterning (BMPs), and cell specification (GATA factors).
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MATERIALS AND METHODS |
DNA and RNA preparation.
cDNAs of human BMP-4,13 Xenopus
GATA-1a,20 Xenopus activin B, and
Xenopus eFGF21 were cloned in the expression
plasmid, pcDNA3 (Invitrogen, Carlsbad, CA), which uses a
cytomegalovirus (CMV) promoter for expression in vertebrate cells. The
GATA-1a plasmid is referred to as pcDNA3-GATA-1 in the rest of the
article. eFGF cDNA was used instead of bFGF cDNA as eFGF has a signal
sequence, unlike bFGF, and has an identical effect as bFGF on animal
caps. pSP64T plasmids containing the dominant negative mouse BMP
receptor ( mTFRII)22 and -galactosidase (-gal) cDNA
were used in an in vitro transcription reaction with SP6 polymerase to
generate capped RNA (Ambion, Austin, TX).
Growth factors.
Recombinant human activin A was obtained from the National Institute of
Diabetes and Digestive and Kidney Diseases (Bethesda, MD) and used at
12 ng/mL. Recombinant human bFGF (Sigma Chemical Company, St Louis,
MO) was used at 200 ng/mL. Recombinant human BMP-4 protein
was used at 1 µg /mL and was a generous gift from Genetics Institute,
Boston, MA.
Embryo injection and animal cap explant culture.
Xenopus laevis embryos were obtained as previously
described23 and staged according to Nieuwkoop and
Faber.24 Embryos for microinjection were placed in
0.5× Marc's Modified Ringers (MMR; 1× MMR = 0.1 mol/L
NaCl, 2 mmol/L KCl, 1 mmol/L MgSO4, 2 mmol/L
CaCl2, 5 mmol/L HEPES, pH 7.8, 0.1 mmol/L EDTA), 3% Ficoll 400 (Sigma). Embryos were injected at the one-cell stage with either
plasmid or synthetic RNA in a volume of 10 nL. The doses of plasmid and
RNA used are described in the figure legends. Control injections used
water or empty pcDNA3 plasmid. At stage 8, animal caps were explanted
from the animal pole of the embryos and cultured for 2 days in cap
culturing solution (0.5× MMR, 0.5 mg/mL bovine serum albumin
(BSA), 50 µg/mL gentamycin, 100 U/mL penicillin, 100 µg/mL
streptomycin), with or without growth factors until sibling whole
embryos reached stage 35 (2 days postfertilization).
Dispersion and reaggregation of animal cap explants.
Animal caps were excised at stage 8 and incubated in
calcium/magnesium-free media (CMFM).25 This caused the
dispersion of the inner cap cells. The pigmented outer layer of the
animal cap was discarded. Dispersed cells from 20 animal caps were
exposed to various growth factors. After 1 to 2 hours of growth factor treatment, the dispersed cells were washed, reaggregated in cap culturing solution, and left to develop until control stage 35.
Isolation of erythroid cells from adult frogs and tadpoles.
Adult erythroid cells were collected in 10 U/mL heparin (Sigma), 0.5%
BSA in 0.7× phosphate-buffered saline (PBS) from an adult frog
wound site. The cells were spread on a slide and stained with Wright
Giemsa. Embryonic erythroid cells were obtained from a stage 41 tadpole
(3 days postfertilization) by cutting off the tail and collecting the
cells that flowed out in a heparinized needle. The cells were
concentrated onto a slide using a Cytospin 3 (Shandon, Pittsburgh,
PA).
Western blot analysis for globin.
Animal caps were suspended in RIPA buffer (10 mmol/L Tris pH 7.5, 5 mmol/L EDTA, 1% Triton X-100, 150 mmol/L NaCl, 0.1% sodium dodecyl
sulfate [SDS], 10% glycerol, 1% aprotinin, 1 mmol/L
phenylmethylsulfonyl fluoride [PMSF]) and incubated at 4°C with
gentle rocking for 15 minutes. Cell debris was removed by
centrifugation. Proteins were separated on a 15% SDS-polyacrylamide
gel and transferred to nitrocellulose (Protran; Schleicher & Schuell,
Keene, NH). One animal cap equivalent was loaded per lane.
The resultant blot was blocked with 5% nonfat milk/Tris buffered
saline + Tween (TBST) for 1 hour at room temperature and
then incubated with a 1:7,500 dilution of a larval  -globin
monoclonal antibody (generous gift from C.H. Katagiri, Hokkaido
University, Japan) in TBST overnight at 4°C.
The primary antibody was detected either by the method of
alkaline phosphatase detection (Promega, Madison, WI; see Fig 6)
or by chemiluminescence (Amersham, Arlington Heights, IL; see Fig 8).
o-Dianisidine staining of cells from whole embryos.
The procedure for detection of hemoglobin in Xenopus was
adapted from a previously described protocol.26 Whole
embryos and animal caps were treated without fixation in a 20:25:1
mixture of 0.14% o-dianisidine in 100% ethanol, 0.2 mol/L
sodium acetate, and 30% hydrogen peroxide for 1 hour. Embryos and caps
were washed with water, fixed in 4% paraformaldehyde, and stored in
methanol. For viewing, the embryos or explants were cleared with benzyl benzoate:benzyl alcohol (1:2).
o-Dianisidine staining of cells from animal caps.
Three animal caps were dispersed in 200 µL of a collagenase B
solution (2 mg/mL in 0.7× PBS). Twenty microliters of an
o-dianisidine solution (10:1 mixture of 0.2%
o-dianisidine [in 0.2% glacial acetic acid] and 30%
hydrogen peroxide) was added to the cell suspension and incubated with
the cells for 1 minute. The cells were pelleted using an Eppendorf
(Madison, WI) centrifuge (4,000 rpm) and the supernatant removed. The
pellets were resuspended in 250 µL of 0.7× PBS, and the cell
suspensions were concentrated onto slides in a Cytospin 3 (Shandon) at
700 rpm for 3 minutes. The slides were fixed in methanol. Orange-brown
color, indicative of globin expression was observed within individual
erythroid cells under light microscopy.
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RESULTS |
Development of a quantitative assay for erythroid cell induction.
To study the induction of hematopoietic mesoderm, we developed a rapid,
robust, and quantitative assay for erythroid cell induction in
Xenopus. This assay uses the totipotent primitive ectoderm from
the animal pole of a midblastula embryo. RNAs or plasmids are injected
into one cell embryos and animal caps excised at stage 8 (Fig 1A). RNAs are translated soon after
injection, whereas plasmids are expressed after the midblastula
transition when zygotic transcription initiates.23 To
evaluate the effect of a particular RNA, plasmid, or growth factor on
the induction of erythroid cells, the treated animal caps are cultured
until control stage 35, when erythroid cells are detected in the VBI of
sibling whole embryos (Fig 2). The cells of
intact animal cap cells are dispersed, stained with the chemical
o-dianisidine, and concentrated onto a glass slide (Fig 1). In
the stage-35 tadpole, only the cells of the VBI stain with
o-dianisidine (Fig 2A), showing that it is a specific and
sensitive indicator of differentiated erythroid cells26
(Fig 2C). The visualization of discretely stained cells by microscopy
allows for quantitation of the inductive assay. In our hands, this
assay is more reproducible than reverse-transcription polymerase chain
reaction (RT-PCR) analysis for globin mRNA expressed by the entire
animal cap. In addition, o-dianisidine staining is indicative
of both globin expression and hemebiosynthesis of erythroid cells.
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| Fig 1.
An assay for the induction of erythroid cells from animal
caps. (A) One-cell stage embryos are injected with RNA or plasmids.
Animal caps are excised at stage 8 and cultured for 2 days under
defined conditions and then dispersed by collagenase. The cells are
incubated with o-dianisidine and immobilized onto a glass slide
for analysis by microscopy. (B) Protein factors can be tested on
dispersed animal caps cells. At stage 8, animal caps are excised and
dispersed in CMFM. The outer pigmented layer is discarded. The
dispersed inner layer cells are incubated with or without factors.
After 1 to 2 hours the cells are washed and reaggregated in
calcium/magnesium containing media and cultured for 2 days.
o-dianisidine cells are detected as described in (A).
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| Fig 2.
o-Dianisidine staining of whole embryos and
erythroid cells. (A) Stage-35 whole embryo; the arrow points to the
blood island that is stained orange-brown. (B) pcDNA3-BMP-4 (200 pg)-
injected embryos; the ventralized embryos have diffuse orange-brown
staining of the mesoderm. The arrow points to the enlarged blood island
of a partially ventralized embryo. (C, D)
o-dianisidine-positive cells from a dispersed stage-33 tadpole
VBI. The cells in (C) were photographed at 10× original
magnification, whereas the cells in (D) were photographed at 20×
original magnification.
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BMP-4 cooperates with mesoderm inducers to generate hematopoietic
mesoderm.
BMP-4 has been thought to initiate the hematopoietic program during
embryogenesis. An embryo ventralized by plasmid BMP-4 injection is
stained with o-dianisidine in a radially symmetric pattern,
corresponding to the expanded blood island (Fig 2B). We used the animal
cap assay to study the role of BMP-4 in hematopoietic induction. BMP-4
RNA (0.5 to 1 ng) and BMP-4 cDNA (200 pg of a CMV-driven BMP-4 plamid,
pcDNA3-BMP-4) were injected into embryos at the one cell stage, and
animal caps were explanted at stage 8 and cultured until control stage
35. Few o-dianisidine-positive cells were detected (plasmid
injection shown in Fig 3A).
The activity of BMP-4 was also evaluated by exposing explanted animal
caps to BMP-4 protein. To allow uniform access of growth factors to the
animal cap cells, the caps were disaggreaged in CMFM at stage 8 and
then treated with BMP-4 protein. After 1 to 2 hours the cells were
washed, reaggregated, and cultured until control stage 35 (Fig 1B). As
shown in Fig 3B, animal cap cells treated with BMP-4 protein alone (1 µg/mL) failed to differentiate into erythroid cells. Thus, even
though forced BMP-4 expression can lead to enlarged ventral blood
island formation12-14 (and this study). BMP-4 is unable to
induce erythroid cells in animal caps. This suggests that there are
additional factors in the embryo that cooperate with BMP-4 to regulate
blood formation.
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| Fig 3.
Erythroid cells are detected in animal caps
treated with BMP-4 and mesoderm inducers. (A) For experiments using
plasmid BMP-4 embryos are injected with control vector or pcDNA3-BMP-4
(200 pg) and treated with activin (12 ng/mL) or FGF (200 ng/mL). (B)
For experiments using BMP-4 protein, animal caps are excised at stage 8 and dispersed in CMFM. The cell dispersion is treated with BMP-4 (1 µg/mL) with and without activin (12 ng/mL) in CMFM for 2 hours before
the cells are washed and allowed to reaggregate in the calcium and
magnesium containing cap culturing solution. In both experiments the
cap cells are cultured until control stage 35 and analyzed for
o-dianisidine-positive cells. The arrowhead points to an
erythroid cell. The black arrow indicates a nonerythroid pigmented
cell. The panels in (A) were photographed at 20× original
magnification and the panels in (B) were photographed at 10× original
magnification.
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We hypothesized that BMP-4 patterns early mesoderm to a ventral fate
and hence hematopoietic fate rather than directly inducing hematopoietic mesoderm. The absent or poor induction of erythroid cells
in animal caps by BMP-4 alone may be due to the absence of direct
mesoderm inducers. To test this hypothesis, pcDNA3-BMP-4-loaded caps,
or dispersed caps incubated with BMP-4 protein, were treated with known
mesoderm inducing factors, such as activin or bFGF (Fig 1A). Control
caps treated with FGF or activin did not induce o-dianisidine
stained cells (Fig 3A). Animal caps loaded with 200 pg of pcDNA3-BMP-4
and treated with activin or bFGF failed to elongate, confirming the
ability of BMP-4 to override the induction of dorsal
mesoderm.13 When these caps were dispersed, significantly higher numbers of cells expressing hemoglobin were evident than that
seen in animal caps injected with pcDNA3-BMP-4 alone (Figs 3A
and 4). On quantitation BMP-4 expression
alone from pcDNA3-BMP4 yielded an average of 87 erythroid cells per
cap, whereas no cells were detected in caps treated with activin or
bFGF alone. BMP-4 plus activin yielded an average of 447 erythroid
cells per cap, whereas BMP-4 plus FGF yielded an average of 536 erythroid cells per cap (averages are determined by counting the number
of cells in six animal caps; Fig 4). In addition, BMP-4 protein (1 µg/mL) added to dispersed cells was able to induce erythroid cells in the presence of activin (Figs 1B and 3B). By Wright-Giemsa staining, the induced cells in the animal caps resemble primitive erythroid cells
(Fig 5). These results show that BMP-4
cooperates with mesoderm inducers to induce erythroid cells.
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| Fig 4.
BMP-4 and activin, or BMP-4 and FGF treatment of animal
caps induce more of o-dianisidine-positive cells than BMP-4
treatment alone. The animal caps were treated as described in Fig 3.
The o-dianisidine-positive cells were counted from two
cytospins for each experimental condition in a representative
experiment. As each cytospin represents three caps, the sum of
o-dianisidine-positive cells from two cytospins was divided by
six to give the number of erythroid cells per cap.
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| Fig 5.
Comparison of morphology of erythroid cells induced in
animal caps to those found in tadpoles and adult frogs. (A) An
erythroid cell from an animal cap treated with BMP-4 and FGF; (B)
circulating erythroid cells in a 3-day-old tadpole (stage 41); (C)
adult erythroid cells. The erythroid cells are stained with Wright
Giemsa. The primitive erythroid cells in (B) are 1 day older than and
look slightly more differentiated than the cell shown in (A). Primitive
erythroid cells are larger and more circular than definitive erythroid
cells; they contain circular and less condensed nuclei and still
possess yolk granules (compare B with C). The panels are photographed
at 40× original magnification.
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We next tested whether the cooperation observed between BMP-4 and
activin, or BMP-4 and FGF, is dependent on the action of activin and
FGF as mesoderm inducers. Animal caps failed to elongate when injected
with CMV-driven activin or FGF plasmids, suggesting that the expression
of activin and FGF from plasmid injections did not induce significant
amounts of mesoderm (data not shown). This is likely to be due to
insufficient accumulation of activin or FGF protein during the period
of competence for mesoderm induction. Furthermore, cooperation between
growth factors generated from CMV-driven activin or FGF and
pcDNA3-BMP-4 was not detected in caps (data not shown). Taken together
with the observation that dispersed animal cap cells differentiate to
erythroid cells with only 1 hour of incubation with BMP-4 and activin
(Fig 3B), these results suggest that FGF and activin act early as
mesoderm inducers.
To determine the dose response of BMP-4 plasmid required for erythroid
induction, larval globin expression was studied by Western blot
analysis of animal caps. The expression of larval globin is indicative
of primitive hematopoietic differentiation. Embryos were injected with
three concentrations of pcDNA3-BMP-4 (5, 20, and 200 pg) and animal
explants treated with or without activin or bFGF
(Fig 6). Activin or bFGF treatment alone
does not induce globin production (Fig 6, lanes 10 and 11). The
induction of globin by pcDNA3-BMP-4 is cooperatively enhanced when the
animal caps are treated with activin or bFGF. Note that in the presence of mesoderm inducers, 20 pg of pcDNA3-BMP-4 induces the same level of
globin protein as 200 pg pcDNA3-BMP-4 (Fig 6, compare lanes 5 and 6 with 8 and 9, respectively). This indicates that 20 pg of pcDNA3-BMP-4
is capable of eliciting maximal globin induction. Doses as low as 0.2 pg pcDNA3-BMP-4 can induce erythroid cells in the presence of mesoderm
inducers (data not shown) and doses of 5 to 20 pg of pcDNA3-BMP-4
induce a high amount of globin protein (Fig 6). The Western blot
analysis is consistent with the o-dianisidine staining results,
suggesting that low levels of BMP-4 RNA can cooperate with mesoderm
inducers to induce erythroid cells.
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| Fig 6.
Dose response of BMP-4 plasmid for globin protein
induction. Western blot analysis for globin expression in animal cap
explants. Embryos were injected with 5, 20, and 200 pg of
pcDNA3-BMP-4, and animal caps were explanted and cultured with or
without activin (12 ng/mL) or bFGF (200 ng/mL) until sibling embryos
were at stage 35. Protein extracts were made from the animal caps and
separated on a SDS polyacrylamide gel. Proteins were transferred to
nitrocellulose and incubated with a larval -globin monoclonal
antibody. Each lane is loaded with one cap equivalence of protein
extract. The finding that 5 pg of pcDNA3-BMP-4-loaded caps treated
with activin (lane 2) induces more globin protein than FGF treatment
(lane 3) is variable.
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GATA-1 can induce erythroid cells without causing ventralization.
BMP-4 is known to induce the expression of the hematopoietic
transcription factor, GATA-1.17 As GATA-1 expression is
downstream of BMP-4 signaling, GATA-1 may specify ventral mesoderm to
become erythroid cells. We tested this hypothesis by incubating
pcDNA3-GATA-1 loaded caps with or without FGF, which has been shown to
participate in ventral mesoderm induction.27 We found that
pcDNA3-GATA-1 injection alone induces low amounts of
o-dianisidine cells, but in the presence of bFGF induces
abundant erythroid cells (Fig 7). Therefore GATA-1 can specify erythroid cells in cooperation with
FGF.
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| Fig 7.
GATA-1 induces erythroid cells in cooperation with bFGF.
(A) Embryos are injected with pcDNA3-GATA-1 (200 pg) and treated with
or without FGF (200 ng/mL). Animal caps are analyzed as described in
Fig 1A. The panels are photographed at 20× original magnification.
The dark cells in the panel of GATA-1 caps treated with no growth
factors are pigmented cells that do not stain with
o-dianisidine.
(B) The o-dianisidine-positive cells
were counted from two cytospins for each experimental condition in a
representative experiment. The number of o-dianisidine cells
per cap was determined as described in Fig 4.
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To determine the dose response of GATA-1 plasmid required for erythroid
induction, embryos were injected with four concentrations of
pcDNA3-GATA-1 (2, 7.5, 20, and 200 pg) and animal caps treated with
bFGF (Fig 8). The animal caps were either
analyzed for globin expression by Western analysis or for hemoglobin by
o-dianisidine staining. Quantitation of
o-dianisidine-positive cells per animal cap induced from each
dose shows that there is erythroid induction at the 2- and 7.5-pg doses
of pcDNA3-GATA-1 (Fig 8B). The o-dianisidine assay is capable
of detecting erythroid induction at levels that are undetected by
Western analysis. This shows the sensitivity of the animal cap assay
using o-dianisidine.
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| Fig 8.
Dose response of GATA-1 plasmid for globin protein and
erythroid cell induction. Embryos were injected with 2, 7.5, 20, and
200 pg of pcDNA3-GATA-1, and animal caps were explanted and cultured
with bFGF (200 ng/mL) until sibling embryos were at stage 35. The
animal caps were analyzed for globin induction by (A) Western analysis
and (B) o-dianisidine staining. The Western analysis was
performed as described in Fig 6. The number of
o-dianisidine-positive cells per cap represents the average of
two cytospins for each dose of pcDNA3-GATA-1 and was determined as
described in Fig 4.
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In contrast to BMP-4 overexpression, GATA-1 overexpression does not
ventralize whole embryos.28 It is possible that GATA-1 could induce erythroid cells in the absence of BMP-4 signaling; however, the animal cap expresses low levels of BMP-4.12 To determine if signaling by this small amount of BMP-4 is required for
GATA-1 function, we used a mutant type I BMP-2/4 receptor (mTFRII)
lacking the serine/threonine kinase domain. The mutant receptor has
been shown to block BMP-4 signaling and ventralization.22 Various doses of mTFRII RNA were coinjected with 200 pg of
pcDNA3-BMP-4 to establish a dose of mutant receptor RNA that would
block induction of erythroid cells by BMP-4. A dose of 1 ng of
mTFRII RNA was found to block the ability of BMP-4 to induce
erythroid cells (Fig 9A and B
[see page 4134]). The injection of 1 ng of mTFRII RNA alone does
not prevent mesoderm from being induced. These animal caps treated with
FGF show elongation, which is an indicator of mesoderm induction (data
not shown). To determine if the mutant receptor could block the
induction of erythroid cells by GATA-1, 200 pg of pcDNA3-GATA-1 with
1 ng of mTFRII RNA was injected into animal caps.
Erythroid cells were not detected (Fig 9C, D, and G), showing that
BMP-4 signaling is involved in the GATA-1 induction of erythroid cells.
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| Fig 9.
GATA-1 requires the BMP pathway to cooperate with bFGF to
induce erythroid cells. Embryos were coinjected with (A) -gal RNA (1 ng) and pcDNA3-BMP-4 (200 pg); (B)  mTFRII RNA (1 ng) and
pcDNA3-BMP-4 (200 pg); (C) -gal RNA (1 ng) and pcDNA3-GATA-1 RNA
(200 pg); (D) mTFRII RNA (1 ng) and pcDNA3-GATA-1 RNA (200 pg); (E)
-gal RNA (1 ng) and control vector (200 pg); and (F) mTFRII RNA
(1 ng) and control vector (200 pg). Animal caps were explanted,
cultured with bFGF (20 ng/mL) until sibling embryos were at stage 35, and analyzed for o-dianisidine-positive cells (Fig 1A). The
panels are photographed at 20× original magnification.
(G) The number of
o-dianisidine-positive cells was counted in two cytospins for
each experimental condition in a single experiment. The number of
o-dianisidine cells per cap was determined as described in Fig
4.
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DISCUSSION |
BMP-4 function in the induction of hematopoietic mesoderm.
BMP-4 acts after the initial steps of mesoderm induction to cause
ventralization and erythroid induction.14,15,17 Our studies
show that BMP-4 cooperates with FGF or activin to induce erythroid
cells in animal cap assay, suggesting that BMP-4 patterns induced
mesoderm to a ventral fate (Figs 3 and 4). Some studies propose that
BMP-4 is a direct ventral mesoderm inducer. This is based on the
detection of mesodermal markers in animal caps loaded with high doses
of BMP-4 RNA.13,19,29-31 The ability of BMP-4 to act as a
patterning molecule or a mesoderm inducer may be determined at the
receptor level. BMP-4 signaling is mediated through the activation of
type I and type II serine/threonine receptors.32,33 Type I
and type II receptors of various TGF- family members are known to
oligomerize upon exposure to particular ligands, which may lead to
qualitatively different signals. For example, the BMPRII receptor can
bind to activinRI (ALK-1) as well as to BMPRIA and
BMPRIB.34 BMP-4 prefers to bind BMPRIA/BMPRII and
BMPRIB/BMPRII to generate a BMP signal. The endogenous levels of BMP-4
in the Xenopus embryo are not known, and it is quite possible
that the level is high on the ventral side of the embryo. High levels
of BMP-4 may force the formation of receptor combinations that generate
an activin signal, which induces mesoderm. This mesoderm could then be
patterned to form erythroid cells by that same high dose of BMP-4.
Thus, although BMP-4 can perform both patterning and inductive
functions in vitro, we believe that it is the patterning activity of
BMP-4 on early mesoderm that results in the induction HSCs.
Activin and/or FGF have been previously postulated to be
important in the induction and differentiation of the erythroid
lineage. Activin was cloned as erythroid differentiation factor
(EDF)35 and can induce in vitro differentiation of
erythroleukemic cell lines at a concentration range of 0.5 to 10 ng/mL.36 Activin (and BMP-4) receptors are expressed on the
erythroid cells of the developing blood island (J. Graff,
personal communication, April 1995). Despite this, activin
predominantly induces dorsal mesoderm in animal caps and is not capable
of inducing globin expression. FGF signaling has been shown to be
required for posterior-ventral mesoderm induction.27 FGF
receptors have been detected in murine bone marrow cells, and bFGF can
be found in the bone marrow extra-cellular matrix37;
however, similar to activin, FGF is insufficient to induce
erythropoiesis in animal caps.27,38,39 Our studies show an
early role for activin and FGF as mesoderm inducers but do not exclude
later roles for these factors during hematopoiesis.
Our studies in Xenopus extend the analysis of BMP-4 function in
higher vertebrates. Targeted gene disruption of the BMP-4 ligand and
type 1 BMP-2/4 receptor in mouse ES cells show an early requirement for
BMP-4 signaling in the early proliferation of the
epiblast.40,41 The most severe phenotype for both ligand- and receptor-deficient embryos is the lack of any mesodermal
derivative, preventing an evalution of the role of BMP-4 signaling in
hematopoietic induction. The BMP-4 mutants that undergo gastrulation
develop with poorly organized posterior structures and a reduction in blood islands.41 These mutants are believed to be partially rescued by BMP-2. Our finding of cooperative effects of BMP-4 and
activin, or BMP-4 and FGF, in the induction of hematopoietic mesoderm
in Xenopus may also exist for higher vertebrates. These combinations may stimulate embryonic stem cell and primary yolk sac
cultures to yield primitive colonies. BMP-4 has some ability to induce
hematopoietic precursors when added to embryonic stem cells,42 and the addition of activin or FGF may lead to
higher numbers of colonies. This would be an advance to studies of
primitive erythroid cells since they are difficult to grow in vitro.
GATA function requires an active BMP cascade.
GATA-1 has been found to regulate most, if not all, erythroid-specific
genes43 and is absolutely required for terminal erythroid differentiation.44,45 In contrast, GATA-2 is required for
the maintenance or proliferation of hematopoietic
progenitors.46 Our studies have shown that GATA-1
cooperates with FGF to induce erythroid cells. In these experiments,
GATA-1 may have a similar activity as GATA-2. GATA-2 overexpression in
animal caps is able to induce globin RNA expression.16
Interestingly, GATA-3, which is involved in thymocyte
development,47 and GATA-4, which participates in heart and
gut,48,49 induce o-dianisidine-positive cells in
our assay (T.H. and Y.Z., unpublished results, July
1996). As GATA factors share a highly conserved zinc
finger-type DNA binding domain, they may activate the same genes when
overexpressed in Xenopus embryos. Our results with GATA-1
therefore point to the involvement of the GATA binding proteins during
HSC specification during embryogenesis.
GATA-1 does not ventralize whole embryos28 and is therefore
not a patterning molecule. It is capable of cooperating with FGF to
induce erythroid cells in animal caps (Fig 7). Based on the injections
of mTFRII, mesoderm that lacks an intact BMP signaling cascade
cannot respond to GATA-1 (Fig 9). A variety of factors such as the
homeobox genes Pv.1, Mix.1, Vent-1, Vent-2/Vox, and Xom are expressed
ventrally in gastrula embryos and in response to BMP-4 in animal
caps.50-55 The genes regulated by these homeobox genes may
be essential for GATA-1 to induce erythroid cells. Understanding the
requirement of BMP-4 and downsteam genes in erythroid induction might
further establish later functions of BMP signaling cascade during
hematopoiesis.
This work suggests a model for hematopoietic stem cell induction during
vertebrate embryogenesis. Mesoderm induction at the late blastula and
early gastrula stage establishes competence of cells to respond to
BMP-4 to form HSCs. The endogenous mesoderm inducers may be FGF,
activin-like molecules, or even BMP-4. The specific role of BMP-4 in
hematopoietic induction is to pattern the mesoderm by affecting the
program of gene expression. Ultimately, hematopoietic transcription
factors, such as the GATA factors, are expressed that mediate the
specification of the HSC fate.
| |
ACKNOWLEDGMENT |
We thank Chris Wright (Vanderbilt University) and Todd Evans (Albert
Einstein College of Medicine) for BMP-4 cDNA, Jonathan Slack
(University of Bath) for eFGF cDNA, Mitsugu Maeno (Niigata University,
Japan) for mTFRII cDNA, and CH Katagiri (Hokkaido University, Japan)
for larval globin monoclonal antibody. BMP-4 protein was a generous
gift from Genetics Institute, Boston, MA. We thank the members of the
laboratory for helpful discussions and suggestions. We thank Drs Jeremy
Green, Xi He, Barry Paw, Sadhana Agarwal, and David Ransom for their
comments on this manuscript. Leonard Zon is an Associate Investigator
of the Howard Hughes Medical Institute.
| |
FOOTNOTES |
Submitted February 24, 1998;
accepted July 30, 1998.
Supported by National Institutes of Health Grant No. 2RO1 HL48801-05
(L.I.Z.).
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 Leonard I. Zon, MD, Division of
Hematology/Oncology, Children's Hospital, 300 Longwood Ave, Enders
650, Boston, MA 02115; e-mail: zon{at}rascal.med.harvard.edu.
| |
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C. J. Marshall, C. Kinnon, and A. J. Thrasher
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Blood,
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[Abstract]
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A. E. Deconinck, P. E. Mead, S. G. Tevosian, J. D. Crispino, S. G. Katz, L. I. Zon, and S. H. Orkin
FOG acts as a repressor of red blood cell development in Xenopus
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S. Robertson, M Kennedy, J. Shannon, and G Keller
A transitional stage in the commitment of mesoderm to hematopoiesis requiring the transcription factor SCL/tal-1
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P Faloon, E Arentson, A Kazarov, C. Deng, C Porcher, S Orkin, and K Choi
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Top
Abstract
Introduction