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First published online 8 December 2005
doi: 10.1242/dev.02192
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Laboratory of Molecular Vertebrate Embryology, The Rockefeller University, New York, NY 10021, USA.
* Author for correspondence (e-mail: brvnlou{at}rockefeller.edu)
Accepted 1 November 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: GDF3, TGFß, BMP, Embryonic stem cell, Inhibitor, Xenopus, Human, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The examination of signaling components specific to the
state of pluripotency revealed a very small number of ligands belonging to the
main signaling pathways involved in early embryogenesis. Notably, only three
secreted factors of the TGFß superfamily are expressed at high levels
during pluripotency and decline dramatically when the cells are allowed to
differentiate, suggesting a role during this process: LEFTY1, LEFTY2 and GDF3
(Sato et al., 2003). Both
LEFTY proteins have been previously shown to be secreted inhibitors of
TGFß ligands (reviewed by Branford and
Yost, 2004). The role of GDF3, however, is unknown, as this
TGFß family member has not been as extensively studied as the others. In
this study, we dissect the cellular and biochemical functions of GDF3 in the
context of embryonic development and ES cells.
GDF3 is a TGFß superfamily member subclassified into the bone
morphogenetic protein/growth and differentiation factor (BMP/GDF) branch of
this family (Jones et al.,
1992). GDF3 is most similar to VG1 (57% amino acid identity, 79%
similarity) and has 53 and 50% identity to BMP2 and BMP4, respectively. GDF3
has only six of the classical seven cysteines present in other TGFß
superfamily members (Jones et al.,
1992). It is missing the fourth cysteine - the one involved in
inter-molecular interactions among TGFß family members and with their
secreted inhibitors (Groppe et al.,
2002).
GDF3 is a mammalian-specific TGFß ligand as it has not been
found in the genomes of fugu, frog, zebrafish or chick. It was first isolated
from a mouse embryonic day (E) 6.5 cDNA embryonic library by homology to
Xenopus VG1, although neither GDF3 mRNA expression nor GDF3 protein
has ever been detected before mid-gestation mouse development
(Jones et al., 1992). Later,
GDF3 is expressed in embryonic bone and adult bone marrow, thymus, spleen and
fat. It is also expressed in the pluripotent mouse teratocarcinoma cell line,
F9, where its expression is reduced upon retinoic acid-induced differentiation
(Jones et al., 1992;
McPherron and Lee, 1993).
Human GDF3 was identified through subtractive hybridization in
activin-treated embryonic carcinoma (EC) cell lines
(Caricasole et al., 1998).
Although very little is known about GDF3, the roles of its other subfamily
members, classic BMPs, have been well characterized. In the early
Xenopus embryo, one of the earliest known functions of BMP signaling
occurs in the late blastula and leads to the inhibition of neural fate in the
dorsal ectoderm and induction of epidermal fate in ventral ectoderm
(Munoz-Sanjuan and Brivanlou,
2002). In mammalian embryogenesis, the first in-vivo cell fate
decision of the blastocyst is between inner cell mass and trophoblast fates.
Recent evidence suggests that BMP signaling also plays a role in this crucial
balance, as the addition of classic BMPs to human ES cells results in
differentiation to trophoblast (Xu et al.,
2002). Conversely, in mouse ES cells, BMPs have been shown to
promote the maintenance of pluripotency, or `stemness', in cooperation with
LIF (Ying et al., 2003).
To study the potential function of GDF3 in the undifferentiated, pluripotent state of ES cells, we characterized GDF3 expression throughout very early murine embryogenesis. We also show that GDF3 is a direct BMP inhibitor in early embryos and pluripotent cells. In addition to its embryological and biochemical characterization, we found that GDF3 acts in human ES cells to maintain markers of pluripotency, while, paradoxically, in mouse ES cells it acts to maintain the ability to differentiate in vitro into cell types representing all germ layers.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and staged as described in Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967).
Embryo explants (animal caps, ventral and dorsal marginal zones) were isolated
in 0.1x MMR, washed once and transferred immediately to 0.5x MMR
with gentamycin for culture. Staging was determined using sibling embryos.
Cell culture
BGN1 and Jasmine human ES cells were maintained as previously described
(Sato et al., 2003). Ten ng/ml
of recombinant human BMP4 (rhBMP4) (R&D) was used for treatments. P19
cells were maintained in MEM-
-modified media (Sigma) with 7.5% CBS,
2.5% FBS. C2C12 cells were maintained in DMEM (Sigma) with 15% FBS. For
differentiation of C2C12 cells, cells were grown to confluence, changed to
DMEM with 2% serum and BMP4 (100 ng/ml) or TGFß1 (R&D) (1 ng/ml) was
added. Genetrap ES cells (The Wellcome Trust Sanger Institute) parent strain
12901a and genetrap AD0857 cells were cultured on gelatin with 1400 U LIF/ml
in 1x GMEM containing 10% FBS, 2 mmol/l glutamine, 1 mmol/l sodium
pyruvate, 1x non-essential amino acids and 55 µmol/l
ß-mercaptoethanol.
Embryoid body formation
Cells were trypsinized to a single cell suspension, counted and diluted to
10,000 cells/ml in ES growth media with no LIF. Droplets of cell suspension
(25 µl) were placed on the lid of a 10-cm bacterial culture dish and
cultured inverted over media for 2 days. Embryoid bodies were then flushed
into EB media (DMEM with 10% FBS) and cultured for 8 days. Percentage EB
formation was assessed by counting the number of embryoid bodies formed/number
of droplets per condition x100%.
RT-PCR
For Xenopus, ten animal caps or one embryo were isolated. For
mouse, five pre-implantation embryos, three gastrulation stage embryos or one
post-gastrulation stage embryo were used. For stem cells, 0.1-1 ng RNA was
used. Primer sequences are available upon request.
Luciferase assays
All luciferase assays were done in three separate experiments, each in
triplicate; representative individual triplicate experiments are shown in the
Results section. In Xenopus embryos, 20 pg of luciferase DNA
construct (BRE-Lux) was injected into the animal region of two cell embryos
together with the indicated RNAs: mouse BMP4 (0.5 ng/embryo), mouse GDF3 (0.5
ng or 1 ng/embryo) transcribed from constructs in pCS2++. Pools of four
embryos were harvested at stage 11 in 50 µl of lysis buffer. In P19 cells,
cells were transfected with 150 ng of reporter, 3.3 ng of renilla reporter,
0.25 µg of SMAD1 and SMAD4 (each), 0.1 µg of OAZ and test constructs in
pCS2++ or empty vector for a total of 1.6 µg/well. After 6 hours of
transfection, media was changed to MEM--modified media with 0.2% serum.
After 32 hours, cells were lysed in 150 µl of lysis buffer and analyzed for
luciferase activity. The error bars indicate standard deviation.
Immunoprecipitations and western blots
Immunoprecipitations on overexpressed proteins were performed as previously
described (Yeo and Whitman,
2001) with mouse FLAG (Sigma; 1 µl/sample) or mouse
HA (Babco; 5 µl/sample). Antibodies used in western blots were mouse
tubulin (Sigma; 1:1000); rabbit cyclophillinB (Affinity
BioReagents; 1:2000); mouse SMAD1 (Santa Cruz; 1:750); mouse
SMAD2 (BD Biosciences; 1:750); rabbit P-SMADs (Cell Signaling;
1:1000); mouse OCT3 (BD Transduction Laboratories; 1:1000); mouse
TROMA1 (Dev Studies Hybridoma Bank; 1:75); goat GDF3 (R&D;
1:10,000); mouse FLAG (Sigma; 1:10,000); mouse HA (Covance;
1:1000). indicates antibody.
In-situ hybridization
Mouse embryos were obtained from wild-type Swiss Webster and C57BL/6
pregnant mice. The day of development was determined as the number of days
following morning observation of a coital plug (E0.5). Embryos were dissected
into cold PBS and fixed, and whole-mount in-situ hybridization was performed
as described (Merrill et al.,
2004). Probes were prepared from Gdf3 in pCS2++
(anti-sense PstI/T3; full-length sense NotI/Sp6). Sectioning
of whole-mount embryos was performed with 10-µm cryosections after
embedding in OCT.
Immunofluorescence
Blastocysts were fixed in 4% paraformaldehyde, then washed in PBS
containing 0.25% BSA, blocked and permeabilized in 10% donkey serum with 3%
Triton, and incubated in primary antibody gGDF3 (R&D) (1:100) in
10% donkey serum for 3 hours at room temperature. Samples were then washed and
incubated in secondary antibody then counterstained with SytoxGreen (Molecular
Probes) for nuclear stain.
Constructs
The coding region of GDF3 was cloned into pCS2++ from pBluescript
(generously provided by S. J. Lee) by PCR and was inserted into the
Eco/Not sites. For sense RNA, GDF3 was linearized with
NotI and transcribed with SP6. BMP4 untagged is in pSP64T and RNA was
produced with AvrII/SP6. BMP4-HA was produced with AvrII/T7.
ActivinB-HA is in pCS2++ and was produced with NotI/SP6. VG1-HA
(provided by S. Cheng) is in pcDNA3.1 and was produced with AvrII/T7.
xNR1 is in pCS2++ and was produced with NotI/SP6.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GDF3 acts as a BMP inhibitor and elicits secondary axis induction and direct neural induction
As our studies in human and mouse ES cells suggested that GDF3 is involved
in the earliest cell fate decisions, we examined the molecular mechanism of
GDF3 activity to determine whether it has classic BMP/GDF signaling activity.
We chose to perform these experiments with microinjection of GDF3 mRNA into
frog embryos, because the effects of TGFß pathway signaling in
Xenopus are well established through phenotype and marker gene
expression analysis. We reasoned that if GDF3 is an agonist or antagonist of
the BMP pathway, its function might be partially determined through its effect
on normal frog embryogenesis. In all frog experiments, mouse GDF3 mRNA was
used, because there is no Xenopus homolog of GDF3. The
Xenopus system is suitable for the study of mammalian proteins, as
all major signaling pathways are conserved, allowing us to study GDF3 function
in a system with no background levels of protein.
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For the reporter assays we used a BMP-responsive promoter element that
drives expression of luciferase (BRE-Lux)
(Hata et al., 2000) in both
Xenopus embryos and P19 cells.
Fig. 2B presents the result of
experiments in which BRE-Lux was co-injected with BMP4, GDF3 or both RNAs.
While BMP4 increased reporter activity, this induction was strongly inhibited
by the presence of GDF3.
To confirm this finding in a cell type that normally expresses GDF3, we
then performed similar experiments in P19 cells into which we transfected
mouse Gdf3 with or without mouse Bmp4. In order to generate
a robust BMP signal, this system was complemented by co-transfection of
downstream signaling components: SMAD1, SMAD4 and OAZ
(Hata et al., 2000).
Fig. 2C shows that in P19
cells, mouse GDF3 reduced BMP4 signaling. This evidence confirms that GDF3 is
an inhibitor of the BMP-GDF subfamily of TGFß in frog embryos and in
mouse pluripotent cells.
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). When
dissected and cultured alone, ectodermal explants of Xenopus blastula
adopted an epidermal fate, and inhibition of the BMP pathway in these cells
led to a direct conversion of epidermal to neural fate. Two-cell embryos were
microinjected in the animal pole with mRNA encoding mouse GDF3 and allowed to
develop to blastula stages. Animal caps were isolated and cultured to either
late gastrula (stage 12.5) or late neurula (stage 21). The explants were then
harvested and analyzed by RT-PCR for the detection of cell-type-specific
markers. Our analysis of expression of early markers
(Fig. 2E) and late markers
(Fig. 2F) shows that, by
contrast to other BMP family members, GDF3 acted as a direct neural inducer.
GDF3 decreased expression of the immediate early response to BMP signaling,
MSX-1, and increased expression of both early (SOX2) and late neural markers,
such as OTX2 and NCAM. This conversion was direct, because it was not
concomitant with mesoderm induction, as demonstrated by the lack of induction
of the pan-mesodermal marker brachyury (Xbra) and the marker of axial
mesoderm, collagen type II. To control for possible artifacts due to microinjection of RNA encoding GDF3, we performed assays with GDF3 protein in cells of the MZ, which are more sensitive to changes in BMP activity. BMPs are normally expressed throughout the early embryo, but the local secretion of BMP inhibitors in the DMZ establishes the dorsal axis in normal Xenopus embryos; therefore, creating a second region of BMP inhibition in the VMZ results in the induction of secondary axial structures.
GDF3 protein was obtained by microinjection of 50 ng GDF3 mRNA into Xenopus oocytes, followed by collection of GDF3-containing medium after 48 hours. We first examined the oocyte lysate and the conditioned media for GDF3 protein to check for processing and secretion of GDF3. In the oocyte lysate, we found both the prepro and mature forms of GDF3, but only the mature form was secreted into the conditioned media (Fig. 2G). The conditioned medium was then presented to both DMZ and VMZ, and the behavior of the explants was compared to controls. Fig. 2H shows that VMZ explants normally formed a sphere in culture, whereas DMZ explants elongated due to the formation of tissues, such as muscle and notochord, that undergo convergent extension. Activin protein can dorsalize VMZ explants (causing elongation), although through a mechanism distinct from BMP inhibition; therefore activin was used as a positive control. GDF3 protein dorsalized VMZ tissue, creating elongation and the appearance of anterior structures such as the cement gland. Analysis of molecular markers by RT-PCR in these explants agreed with the conclusion that GDF3 has dorsalizing activity in the mesoderm, as GDF3 protein decreased the expression of the ventral mesodermal marker globin and induced the expression of dorsal paraxial markers such as muscle actin in the VMZ explants (see Fig. S1 in the supplementary material). These data collectively demonstrated that GDF3 acts as a BMP inhibitor in both frog embryos and in pluripotent mammalian cells; however, its mechanism of BMP inhibition remained unknown.
Mechanism of GDF3 action
BMP inhibition may occur at the level of secretion of BMP ligands, receptor
binding, SMAD phosphorylation, transcriptional activation or crosstalk with
other pathways. As GDF3 is a secreted ligand, and because GDF3 mature protein
can function when presented to cells in solution and is expressed in regions
of the embryo that do not colocalize with BMP expression (see below), we
hypothesized that GDF3 acts as an extracellular inhibitor. To determine
whether GDF3 inhibition of BMPs is direct, we performed reciprocal
coimmunoprecipitation assays and found that GDF3 and BMP4 protein interact
(Fig. 3A). HA-tagged BMP4 (or
untagged BMP4) and FLAG-tagged GDF3 were injected into the animal poles of
frog embryos at the 2- to 4-cell stage, and embryos were cultured to gastrula
stage. The animal caps were lysed and immunoprecipitated using anti-HA or
anti-FLAG. FLAG-GDF3 immunoprecipitated prepro and mature BMP4 (60 and 26 kDa)
and HA-BMP4 immunoprecipitated the prepro (45 kDa) and mature forms (18 kDa)
(Fig. 3A). Similar results were
obtained with FLAG-GDF3 immunoprecipitation of untagged BMP4 (data not
shown).
We also tested whether GDF3 interacts with other TGFß members to determine whether the interaction between GDF3 and BMP4 is specific or reflective of promiscuous binding by GDF3, and we found that GDF3 did not interact with activin (Fig. 3B). Interestingly, although the prepro form is the major form produced in cells and immunoprecipitated in these assays, GDF3/BMP interactions do not rely on the prepro domain. We tested this by using GDF3 to immunoprecipitate BMP4 containing the BMP4 prodomain or the activin pro-domain and found that GDF3 immunoprecipitated both forms of BMP4, whereas it did not interact with activin (data not shown).
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GDF3 partially maintains pluripotent cell types in human ES cells
It has previously been shown that exogenous BMP signaling from other
BMP/GDFs promotes extra-embryonic cell fate differentiation in human ES cells
(Xu et al., 2002). To
determine whether, as a BMP inhibitor, GDF3 can oppose these functions, we
transiently transfected BGN1 or Jasmine human ES cells with Gdf3
plasmids and cultured these cells in CM, which maintains the undifferentiated
state, or in the absence of CM, which allows heterogeneous differentiation,
and in the presence or absence of BMP4 protein.
Fig. 4A shows that
Gdf3-transfected BGN1 human ES cells maintained significant levels of
the pluripotency markers OCT3/4 and NANOG in conditions that normally promote
differentiation but did not have increased levels of TROMA1, a marker of
trophoblast (data not shown). In Jasmine human ES cells, we found that
GDF3 overexpression resulted in a more limited maintenance of
pluripotency markers upon differentiation, confined to the combined treatment
of BMP4 protein and the absence of conditioned medium
(Fig. 4B). In addition,
GDF3 overexpressing cells had a more compact, stem-like morphology,
whether cultured in the presence of CM or in differentiating conditions (data
not shown). This demonstrates that GDF3 contributed to the maintenance of
pluripotent gene expression in human ES cells.
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). To analyze what required functions GDF3 has in mouse ES
cells, we sought to perform reduction-of-function experiments and used ES
cells containing the ß-geo genetrap AD0857, which interrupts the GDF3
gene after exon 1 (and parent strain 129Ola), available from the Sanger
Institute GeneTrap Resource. As shown in
Fig. 5A, exon 1 of GDF3
contains approximately the first third of the prepro domain (red). Exon 2
contains the remaining two-thirds of the prepro domain and all of the mature
domain (green). In the AD0857 genetrap, exon 1 is fused to ß-geo
(Fig. 5A), and in these ES
cells, GDF3 prepro and mature protein were reduced significantly
(Fig. 5B). When cultured in the presence of LIF, wild-type mouse ES cells could be maintained in the undifferentiated state, but in the absence of LIF, these cells differentiated to a flattened morphology after 4 days of culture. By contrast, AD0857 genetrap cells maintained a normal, undifferentiated morphology even in the absence of LIF (Fig. 5C). We analyzed these cells with molecular markers to determine what cell fates are formed and found that, in the absence of LIF, wild-type cells expressed high levels of brachyury, a mesoderm marker, and low levels of OCT3/4 and SOX2, markers of the undifferentiated state, and of FGF5, a marker of pluripotent epiblast; AD0857 cells had reduced levels of brachyury and maintained significant levels of OCT3/4, SOX2 and FGF5 (Fig. 5D). These findings suggest that the reduced levels of GDF3 protein in AD0857 cells precludes normal differentiation.
We tested the ability of AD0857 cells to remain functionally pluripotent even in the absence of LIF by assaying for the formation of embryoid bodies (EBs). Upon culture in hanging drops, undifferentiated ES cells form aggregates, EBs, that differentiate into many types of embryonic tissue. Wild-type and AD0857 cells grown in the presence of LIF formed EBs in 100% of the hanging drops (±0%). While wild-type cells grown in the absence of LIF rarely formed EBs (6±13%), AD0857 cells without LIF formed EBs in 79% (±25%) of the hanging drops (Fig. 5E). However, these `EBs' were much smaller and less compact than EBs produced by cells grown in LIF (Fig. 5F).
We examined the EBs on day 8 of suspension culture by RT-PCR to determine whether a reduction of GDF3 levels alters cell fate outcomes in differentiated mouse ES cells. We studied mRNA levels of stem/primitive markers (OCT4, FGF5, SOX2), neural markers (SOX2, PAX6, RAX), mesodermal markers (NKX2.5, FLK1, SCL1) and endodermal markers (AFP, HNF3ß) (data not shown) (Fig. 5G). While wild-type or AD0857 cells grown in the presence of LIF can give rise to a full profile of differentiated cells types, AD0857 cells grown in the absence of LIF retain a primitive phenotype with some neural differentiation. These cells do not form mesoderm or endoderm and do not express all neural markers tested. This evidence establishes that wild-type levels of GDF3 activity are required for normal in-vitro differentiation of the three embryonic germ layers: the signature of pluripotency.
We next sought to determine whether reduction of GDF3 levels also precludes normal differentiation in vivo by injecting stable GFP-expressing wild-type or AD0857 genetrap ES cells cultured in the presence or absence of LIF into mouse blastocysts and assessing tissue contribution at mid-gestation (E9.5). In this assay of ES cell potential, we found that neither wild-type nor AD0857 ES cells cultured without LIF gave rise to any differentiated cells within the host embryo (data not shown). Further, we did not observe any difference in tissue contribution between wild-type and AD0857 genetrap ES cultured in the presence of LIF, as both differentiated normally (data not shown). These data suggest that the effects of reduced GDF3 levels on ES cell potential are confined to in-vitro differentiation.
GDF3 expression
We analyzed the expression of GDF3 during early mouse embryogenesis to gain
a greater understanding of the role it may play in development. Temporal
analysis of GDF3 mRNA expression by RT-PCR revealed that GDF3 was present at
blastocyst and gastrula stages, strongly upregulated at E8.5, and
significantly reduced by E10.5 (Fig.
6A). Using in-situ hybridization to localize GDF3 expression, we
found that GDF3 was expressed in the blastocyst embryo within the inner cell
mass, from which ES cells are derived (data not shown). However, we could not
exclude staining in the trophoblast cells of the blastocyst, so we performed
immunofluorescence to localize GDF3 protein. We found that GDF3 protein is
expressed throughout the blastocyst embryo and is either membrane-associated
or extracellular (Fig. 6B).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expression of GDF3 in multipotent cell types such as ES cells, neural
crest and teratomas suggests that GDF3 has a significant role in pluripotency
(Sato et al., 2003;
Ramalho-Santos et al., 2002).
Being expressed in the blastocyst, GDF3 is therefore one of the earliest BMP
inhibitors expressed in the mammalian embryo. In fact, recent studies
profiling genome-wide expression in early mouse embryos describe expression of
only one other possible BMP inhibitor, LEFTY, in preimplantation mouse
development (in addition to GDF3) (Hamatani
et al., 2004; Wang et al.,
2004). Later in development, GDF3 is expressed in the node, a
region known to be associated with BMP inhibition, and in the ventral neural
tube. As BMP signaling from the roof plate promotes the dorsalization of
neural cell types (Liem et al.,
1997), GDF3 in the ventral neural tube may function to restrict
these effects.
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; Pera et al.,
2004) while promoting the undifferentiated state in mouse ES cells
(Ying et al., 2003;
Qi et al., 2004). We present
three possibilities to explain these phenomena.
First, it is possible that distinct signaling profiles regulate the early
cell fate decisions of human and mouse embryos. Second, it is possible that
human and mouse ES cells correspond to distinct stages of in-vivo development,
as human ES cells can form trophoblast (the first cell fate decision in the
embryo) while mouse ES cells do not, but upon differentiation can form
primitive endoderm (the second cell fate decision in the embryo). A third
possibility is that human and mouse ES cells possess different sensitivities
to BMP signaling and that different levels of effective BMP signaling in these
cells produces their disparate phenotypes. In support of this model, BMPs can
act as morphogens, creating distinct cell fates based on different
concentrations of the ligand in a given time window
(Wilson et al., 1997). Thus,
it is probable that GDF3 expression helps to establish a BMP activity
gradient. Although its activities may be species-specific, GDF3 regulates both
the ability of stem cells to maintain the undifferentiated state and the
ability to differentiate to the full spectrum of cell types.
Despite the fact that our genetrap experiments provided a reduction, not a loss, of GDF3 function, we detected a dramatic outcome on the state of in-vitro pluripotency of mouse ES cells, as shown by the qualitative and quantitative differences displayed by the embryoid bodies. This observation highlights the critical importance of the regulation of thresholds of BMP activity in the establishment of discrete fates in the mammalian embryo. The role of BMP morphogens as instructive signals in the establishment of early embryonic cell fates is therefore maintained throughout evolution from Drosophila to humans. As each threshold of BMP activity can lead to a completely different cell fate outcome, we expect that a complete loss of GDF3 function (in progress) will elicit a distinct result.
While we have shown that GDF3 inhibits BMP signaling, we cannot rule out
that GDF3 has additional functions, either as an inhibitor or as a ligand. For
instance, GDF3 shares the distinct lack of the fourth cysteine with GDF9, a
TGFß ligand that has been shown to act as a SMAD2/3 activator
(Mazerbourg et al., 2004). In
support of this possibility, we found that very high levels of mouse GDF3 mRNA
injection into frog embryos produced an activin-like response.
We have previously shown that inhibition of GSK3 through BIO, a small
molecule, is sufficient to maintain stemness
(Sato et al., 2004), and here
we show that GDF3 also plays an important role in both mouse and human ES
cells. BIO maintains the expression of GDF3, which we show regulates
thresholds of BMP activity. This represents the beginning of our understanding
of the signaling network involved in the maintenance of the basic aspects of
pluripotency in ES cells. For this picture to be complete, the pathways that
mediate the pluripotency of the inner cell mass and other stem cell types in
vivo must be determined to appreciate the relationship between primitive cell
fates and how they differentiate to create the entire repertoire of cells in
an organism.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/2/209/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Branford, W. W. and Yost, H. J. (2004). Nodal signaling: CrypticLefty mechanisms of antagonism decoded. Curr. Biol. 14,R341 -R343.[CrossRef][Medline]
Caricasole, A., van Schaik, R., Zeinstra, L. M., Wierikx, C. D., van Gurp, R., van den Pol, M., Looijenga, L., Oosterhuis, J. W., Pera, M. F., Ward, A. et al. (1998). Human growth-differentiation factor 3 (hGDF3): developmental regulation in human teratocarcinoma cell lines and expression in primary testicular germ cell tumors. Oncogene 16,95 -103.[CrossRef][Medline]
Groppe, J., Greenwald, J., Wiater, E., Rodriguez-Leon, J., Economides, A. N., Kwiatkowski, W., Affolter, M., Vale, W. W., Belmonte, J. C. and Choe, S. (2002). Structural basis of BMP signalling inhibition by the cystine knot protein Noggin. Nature 420,636 -642.[CrossRef][Medline]
Hamatani, T., Carter, M. G., Sharov, A. A. and Ko, M. S. (2004). Dynamics of global gene expression changes during mouse preimplantation development. Dev. Cell 6, 117-131.[CrossRef][Medline]
Hata, A., Seoane, J., Lagna, G., Montalvo, E., Hemmati-Brivanlou, A. and Massague, J. (2000). OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways. Cell 100,229 -240.[CrossRef][Medline]
Hemmati-Brivanlou, A. (1999). Xenopus. In Encyclopedia of Molecular Biology (ed. T. E. Creighton), pp. 2793-2803. New York: John Wiley and Sons.
Jones, C. M., Simon-Chazottes, D., Guenet, J. L. and Hogan, B.
L. M. (1992). Isolation of Vgr-2, a novel member of the
transforming growth factor-ß-related gene family. Mol.
Endocrinol. 6,1961
-1968.
Liem, K. F., Tremml, G. and Jessell, T. M. (1997). A role for the roof plate and its resident TGFß-related proteins in neuronal patterning in the dorsal spinal cord. Cell 91,127 -138.[CrossRef][Medline]
Mazerbourg, S., Klein, C., Roh, J., Kaivo-Oja, N., Mottershead,
D. G., Korchynskyi, O., Ritvos, O. and Hsueh, A. J. W.
(2004). Growth differentiation factor-9 signaling is mediated by
the type 1 receptor, activin receptor-like kinase 5. Mol.
Endocrinol. 18,653
-665.
McPherron, A. C. and Lee, S. J. (1993). GDF-3
and GDF-9: Two new members of the transforming growth factor-ß
superfamily containing a novel pattern of cysteines. J. Biol.
Chem. 268,3444
-3449.
Merrill, B. J., Pasolli, H. A., Polak, L., Rendl, M.,
Garcia-Garcia, M. J., Anderson, K. V. and Fuchs, E. (2004).
Tcf3: a transcriptional regulator of axis induction in the early embryo.
Development 131,263
-274.
Munoz-Sanjuan, I. and Brivanlou, A. H. (2002). Neural induction, the default model, and embryonic stem cells. Nat. Rev. Neurosci. 3,271 -280.[CrossRef][Medline]
Nieuwkoop, P. D. and Faber, J. (1967).A Normal Table of Xenopus Development (Daudin) . Amsterdam: North-Holland Publishing Company.
Pera, M. F., Andrade, J., Houssami, S., Reubinoff, B., Trounson,
A., Stanley, E. G., Oostwaard, D. W. and Mummery, C. (2004).
Regulation of human embryonic stem cell differentiation by BMP-2 and its
antagonist noggin. J. Cell Sci.
117,1269
-1280.
Qi, X., Li, T. G., Hao, J., Hu, J., Wang, J., Simmons, H.,
Miura, S., Mishina, Y. and Zhao, G. Q. (2004). BMP4 supports
self-renewal of embryonic stem cells by inhibiting mitogen-activated protein
kinase pathways. PNAS
101,6027
-6032.
Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R. C. and
Melton, D. A. (2002). `Stemness': transcriptional profiling
of embryonic and adult stem cells. Science
298,597
-600.
Sato, N., Sanjuan, I. M., Heke, M., Uchida, M., Naef, F. and Brivanlou, A. H. (2003). Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev. Biol. 260,404 -413.[CrossRef][Medline]
Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. and Brivanlou, A. H. (2004). Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55-63.[CrossRef][Medline]
Wang, Q. T., Piotrowska, K., Ciemerych, M. A., Milenkovic, L., Scott, M. P., Davis, R. W. and Zernicka-Goetz, M. (2004). A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo. Dev. Cell. 6, 133-144.[CrossRef][Medline]
Wilson, P. A., Lagna, G., Suzuki, A. and Hemmati-Brivanlou, A. (1997). Concentration-dependent patterning of the Xenopus ectoderm by BMP4 and its signal transducer Smad1. Development 124,3177 -3184.[Abstract]
Xu, R. H., Chen, X., Li, D. S., Li, R., Addicks, G. C., Glennon, C., Zwaka, T. P. and Thomson, J. A. (2002). BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20,1261 -1264.[CrossRef][Medline]
Yeo, C. Y. and Whitman, M. (2001). Nodal signals to smads through cripto-dependent and cripto-independent mechanisms. Mol. Cell 7,949 -957.[CrossRef][Medline]
Ying, Q. L., Nichols, J., Chambers, I. and Smith, A. (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115,281 -292.[CrossRef][Medline]
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