Download An FGF4-FRS2[alpha]-Cdx2 Axis in Trophoblast Stem Cells Induces

TISSUE-SPECIFIC STEM CELLS
An FGF4-FRS2a-Cdx2 Axis in Trophoblast Stem Cells Induces
Bmp4 to Regulate Proper Growth of Early Mouse Embryos
MICHIKO MUROHASHI,a,b TAKAHISA NAKAMURA,c SATOSHI TANAKA,e TAEKO ICHISE,d NOBUAKI YOSHIDA,d
TADASHI YAMAMOTO,c MASABUMI SHIBUYA,a JOSEPH SCHLESSINGER,f NORIKO GOTOHa,b
Division of Genetics, University of Tokyo, Tokyo, Japan; bDivision of Systems Biomedical Technology,
University of Tokyo, Tokyo, Japan; cDivision of Oncology, University of Tokyo, Tokyo, Japan; dGene Expression
and Regulation, Institute of Medical Science, University of Tokyo, Tokyo, Japan; eLaboratory of Cellular
Biochemistry, Animal Resource Science/Veterinary Medical Science, University of Tokyo, Tokyo, Japan;
f
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA
a
Key Words. Trophoblast stem cells • Cdx2 • Bmp4 • FRS2a • Trophectoderm • Stem cell niche
ABSTRACT
A variety of stem cells are controlled by the actions of multiple growth factors in vitro. However, it remains largely
unclear how growth factors control the proliferation and
differentiation of stem cells in vivo. Here, we describe a
novel paracrine mechanism for regulating a stem cell niche
in early mammalian embryos, which involves communication between the inner cell mass (ICM) and the trophectoderm, from which embryonic stem (ES) cells and
trophoblast stem (TS) cells can be derived, respectively. It is
known that ES cells produce fibroblast growth factor
(FGF)4 and that TS cells produce bone morphogenetic pro-
tein (Bmp)4. We provide evidence that FRS2a mediates
activation of the extracellular signal-regulated progein kinase (ERK) pathway to enhance expression of transcription
factor Cdx2 in TS cells in response to FGF4. Cdx2 in turn
binds to an FGF4-responsive enhancer element of the promoter region of Bmp4, leading to production and secretion
of Bmp4. Moreover, exogenous Bmp4 is able to rescue the
defective growth of Frs2a-null ICM. These findings suggest
an important role of Cdx2 for production of Bmp4 in TS
cells to promote the proper growth of early mouse embryos.
STEM CELLS 2010;28:113–121
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
There is an increasing interest in stem cells as potential therapeutic reagents for various degenerative diseases and damaged
organs. In fact, a variety of multipotent stem cells have
recently been discovered in many tissues and organs and
much effort has been made to manipulate them in vitro by
using growth factors, such as FGFs and bone morphogenetic
protein (Bmp)s. However, it is often difficult to expand selfrenewing stem cells or progenitor cells efficiently and differentiate them into specific lineage-committed cell types in
vitro. One of the reasons for this is that it remains largely
obscure how growth factors regulate stem cells and their
niches, the microenvironment surrounding stem cells, in vivo.
Here, we describe one of the molecular mechanisms for the
regulation of stem cells and their niche in mouse early
embryos as a model system.
The trophectoderm (TE) and the ICM in blastocysts contain the first two differentiated lineages of cells during the development of mammalian embryos. The ICM, from which ES
cells can be derived, gives rise to the epiblast and finally the
entire fetus. The TE is the outer epithelial layer of the blastocyst and contributes to the placenta, which is composed of tissues of trophoblast lineages. TS cells can be derived from the
TE [embryonic day (E) 3.5], the extraembryonic ectoderm
(ExE) (E 6.5), and the chorionic ectoderm (E 7.5) [1].
Although TS cells self-renew in vitro in the presence of
FGF4, which is produced by the ICM in vivo [2, 3], they differentiate into various trophoblastic subtypes in the absence
of FGF4. It is thus hypothesized that FGF4 provides a niche
for TS cells in embryos [4]. The polar TE, but not the mural
TE, seems to maintain TS cells because it overlies the ICM
and is accessible to FGF4. At present, the molecular mechanisms regulating the stem cell niche are not well understood.
FGF signaling plays a critical role in the early stages of
embryogenesis [3, 5–10]. Fgf4-null embryos become arrested
in their development around the time of implantation [5].
They undergo uterine implantation and induce uterine decidualization but do not continue to develop. FGF4-deficient blastocysts display a severely impaired proliferation of the ICM
in vitro. It is also reported that postimplantation lethality of
Author contributions: M.M.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript
writing; T.N., S.T., and T.I.: provision of study material; N.Y. and T.Y.: administrative support; M.S.: administrative support, financial
support; J.S.: provision of study material or patients, final approval of manuscript; N.G.: conception and design, manuscript writing, final
approval of manuscript, financial support.
Correspondence: Noriko Gotoh, M.D., Ph.D., Division of Systems Biomedical Technology, Institute of Medical Science, University of
Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan. Telephone: þ81-3-5449-5629; Fax: þ81-3-5449-5425; e-mail: ngotoh@
ims.u-tokyo.ac.jp Received May 14, 2009; accepted for publication October 24, 2009; first published online in STEM CELLS EXPRESS
C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.247
November 3, 2009. V
STEM CELLS 2010;28:113–121 www.StemCells.com
114
Fgf4-null embryos likely results from the failure of proper
differentiation and functions of extraembryonic cell types
[11]. The phenotype of mice with a deletion of Exon 9 of
Fgfr2 resembles that of Fgf4-null mice [3].
The membrane-linked docking protein FRS2a is a major
mediator of FGF signaling [12–14]. Upon stimulation with
FGFs, FRS2a becomes tyrosine-phosphorylated and creates
two specific binding sites for Shp2, an SH2 domain-containing tyrosine phosphatase, and four binding sites for Grb2, an
adaptor protein that links a variety of receptor tyrosine kinases to the Ras/ERK cascade [15, 16]. FRS2a plays critical
roles in the activation of ERK and phosphatidyl inositol (PI)3 kinase in response to FGF and is required for a variety of
FGF-induced developmental processes [15, 17, 18]. The
Shp2-binding sites in FRS2a have a primary role in the activation of ERK whereas the Grb2-binding sites have a primary
role in the activation of PI-3 kinases and a secondary role in
the activation of ERK [16]. Frs2a begins to be expressed in
the early stages of development. Compared with its weak
expression in the epiblast, Frs2a is strongly expressed in ExE
and TS cells [15, 19]. We have previously shown that Frs2anull mice die shortly after implantation caused by the defect
to maintain TS cells [15]. Frs2a-null blastocysts attach to the
dishes, but fail to develop TS cell colonies even in the presence of FGF4. TS cell lines could not be established from
Frs2a-null blastocysts. We also have previously shown that
activation of ERK and expression of Bmp4 are reduced in the
Frs2a-null embryos [15]. Moreover, we showed that FGF4
rapidly induces expression of Bmp4 in wild-type TS cells.
Bmp4 is a member of the TGF-b superfamily and is crucial to the proliferation and differentiation of many different
tissues and organs [20, 21]. Bmp4 stimulates the phosphorylation of Smad1/5/8, which is then translocated to the nucleus
to regulate the transcription of various genes. Bmp4 is
expressed in the polar TE, ExE, and extraembryonic mesoderm [15, 22, 23]. It is reported that Bmp4 is required for development of the primordial germ cells and allantois, both of
them arising from the proximal epiblast [22, 24]. Although
embryos undergo left-right patterning, the ExE-derived Bmp4
has an essential role in the normal formation of the node and
primitive streak [25]. As for the regulation of Bmp4 expression, previous reports suggest that the following transcription
factors are involved in transcription: USF in a mouse
osteoblastic cell line [26], COUP-TF I in fetal rat calvarial
osteoblasts [27], and AP-1 (c-Jun) in Xenopus embryo [28].
However, the molecular mechanisms underlying the developmentally regulated expression of Bmp4 are still unclear.
It has been reported that several transcription factors, such
as Cdx2, are specifically expressed in TS cells but not in differentiated trophoblast cells upon withdrawal of FGF4. Cdx2 is a
member of the caudal-related homeobox transcription factors
[29]. Cdx2 has been reported to be specifically expressed in the
TE at the blastocyst stage and its expression is maintained
within the proliferating ExE [30]. Homozygous Cdx2 mutants
fail to form trophoblast giant cells or produce TS cell lines in
vitro because the TE fails to develop and the mutant embryos
cannot implant into the uterus [30]. It was shown that Cdx2
plays an important role in the development of the TE lineage
cells from totipotent ES cells by forming a complex with Oct3/
4 in to repress Oct3/4’s target genes in ES cells [31]. It has also
been reported that oncogenic Ras/ERK pathway upregulates
Cdx2 expression in ES cells and promotes formation of the TE
[32]. However, no genes under the control of Cdx2 in the TE or
TS cells have yet been identified [12].
In this study, we first analyzed the molecular mechanisms
by which FGF4 induces expression of Bmp4 in TS cells. We
found that the 50 -flanking region of Bmp4 has enhancer activ-
Cdx2 Regulates Bmp4 Expression in TS Cells
ity in response to FGF4 and that this activity can be ascribed
to the binding of Cdx2 to the enhancer element. Furthermore,
expression of Cdx2 and Bmp4 in TS cells was found to be
dependent on activation of an FGF4-induced MEK-ERK pathway. These results indicate that Cdx2 is a key transcription
factor for induction of Bmp4 expression in TS cells. Finally,
using Frs2a-null embryos, our findings suggest that FRS2amediated production of Bmp4 is important for proper growth
of the ICM and epiblast cells.
MATERIALS
AND
METHODS
Luciferase Assay
The 50 -flanking region and introns of Bmp4 used in this study
were a gift from Dr. T. Kurihara. The 50 -1.4 kb (2321 bp to
949 bp), 50 -2.3 kb (2328 bp to 106 bp), intron 1A (þ288 bp
to þ2158 bp), and intron 1B (þ2259 bp to þ3227 bp) [33]
sequences were inserted into the luciferase vector PGVB2 (Toyo
Ink Group, Tokyo, http://www.toyoink.com). The deletion vectors
were constructed based on the ‘‘50 -1.4 kb’’ vector. TS cells were
transiently transfected with each test plasmid DNA, pRL-TK
(Toyo Ink Group), and pcDNA by using LipofectAMINE 2000
(Invitrogen, Carlsbad, CA, http://www.invitrogen.com) with or
without FGF4 (20 ng/ml). Cells were then treated with 10 lM of
an inhibitor, U0126, SB202190, or LY294002 (Calbiochem, San
Diego, http://www.emdbiosciences.com) after 4 hours and harvested after 24 hours.
TS Cell Culture
The cells were cultured as previously described [2]. In brief, TS
cells were cultured by TS medium [2] (Materials and Methods in
the supporting information) with 25 ng/ml FGF4 and 1 lg/ml
Heparin supplemented with 70% mouse embryonic fibroblast-conditioned medium.
Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extracts were prepared from TS cells cultured with or without FGF4 for 48 hours. The Bmp4-wild type (wt) probe (50 -TGGGC
CAAAGGTCACTTTATTGTCTGGGTTTCAGCAAAAT-30 ) corresponding to 2110 bp to 2070 bp of the mouse Bmp4 gene [34].
The Bmp4-mutant (mut) probe has mutations at the Cdx2 binding
site
of
the
Bmp4-wt
probe
(50 -TGGGCCAAAGGTCACGCGTGTGTCTGGG TTTCAGCAAAAT). EMSA was performed with an EMSA kit (Pierce, Rockford, IL, http://www.piercenet.com). Twenty femtomoles of 30 -biotin labeled oligonucleotide
and the nuclear extract (3 lg) were used with or without a 200-fold
excess of unlabeled Bmp4-wt, MUC2-WT probe [29], Bmp4-mut
or MUC2-Mut probe [29], or 1 ll of an anti-CDX2 antibody
(BioGenex, San Ramon, CA, http://www.biogenex.com).
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) analyses were performed
using a ChIP assay kit (Millipore, Billerica, MA, http://www.millipore.com) according to the manufacturer’s instructions. Further
information is described in Materials and Methods in the supporting information.
siRNA Transfection
siRNA was transfected using LipofectAMINE RNAiMAX (Invitrogen) according to the manufacturer’s instructions. The siRNA
sequences are as follows: siCdx2-wt: 50 -CAAGGACGUGAG
CAUGUAUCC; Scramble: 50 -CAAGGACGUGCUAAUGUAUCC.
Transfected TS cells were cultured for 36 hours before the withdrawal of FGF4.
Murohashi, Nakamura, Tanaka et al.
115
Figure 1. Transcriptional activity of Bmp4 promoters stimulated by FGF4 in TS cells. TS cells were transfected transiently with the indicated
constructs and treated with or without FGF4. The results of transcriptional activity measurements were obtained from at least three separate transfections and indicated as the mean 6 SD. (A; left): Schematic representation of luciferase reporters containing mouse Bmp4 promoter regions
indicated by lines. (B, C; left): Deletion vectors were constructed based on the 50 -1.4 kb vector. (D): EMSA. The biotin-labeled Bmp4-wt probe
alone was loaded in lane 1. Nuclear extracts of TS cells cultured with FGF4 (lanes 2-7) or without FGF4 (lanes 8 and 9) were incubated with the
biotin-labeled Bmp4-wt probe (lanes 2-9), in the presence of anti-CDX2 antibody (lanes 3 and 9) or a 400-fold excess of unlabeled Bmp4-wt
probe (B4w) (lane 4) or MUC2-WT (MUw), which have the known Cdx2-binding site (S) (lane 6), or Bmp4-mut probe (B4m) (lane 4) or
MUC2-Mut (MUm), which has mutations at the Cdx2-binding sites of MUC2-WT (lane 7). Nonspecific bands are indicated by a black arrow.
(E): Chromatin immunoprecipitation assays were performed with the indicated antibodies using FGF4-stimulated and unstimulated TS cell
lysates. Abbreviations: FGF, fibroblast growth factor; TS, trophoblast stem.
Immunoblot
TS cells were treated with inhibitors (10 lM U0126, SB202190,
or LY294002) for 1 hour and stimulated with 50 ng/ml FGF4 for
15 minutes or 24 hours after starvation for 6 hours. Anti-Bmp4
(R&D Systems Inc., Minneapolis, http://www.rndsystems.com),
anti-ERK1/2 (Cell Signaling Technology, Beverly, MA, http://
www.cellsignal.com), and anti-phospho ERK1/2 (Cell Signaling
Technology), anti-Akt (Cell Signaling Technology), anti-phospho
Akt (Ser 473) (Cell Signaling Technology), and anti-CDX2 antibody were used. The secretion of Bmp4 was examined with 2.5%
fetal bovine serum TS medium.
Animals
Animals were handled according to the guidelines of the Institute
of Medical Science, University of Tokyo. The experiments were
approved by the committee for animal research at the institution.
Blastocyst Culture
Frs2a/þ mice in the Swiss Webster background were crossed
for production of mutant embryos as previously described [15,
16]. Blastocysts (E 3.5) were cultured for 4 days with TS me-
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dium. After 1 and 3 days, 100 ng/ml recombinant BMP-4 (R&D
Systems, Inc.) was added to the Frs2a/þ crossed blastocysts or
500 ng/ml recombinant Noggin (R&D Systems, Inc.) was added
to Swiss Webster and BL6/J crossed wild-type blastocysts. Diameters or areas of the TE and ICM were measured on the images
after taking photos of 4-day-cultured embryos. Genotyping was
performed as described previously [15], using the TE part of the
embryos.
Alkaline Phosphatase Staining
Alkaline phosphatase staining was performed using an alkaline
phosphatase staining kit (Sigma-Aldrich, St. Louis, http://www.
sigmaaldrich.com) according to the manufacturer’s protocol.
Immunofluorescence
Cultured blastocysts were fixed, permeabilized by 4% paraformaldehyde/0.2% Triton X/phosphate-buffered saline, blocked by
a blocking reagent (DAKO, Glostrup, Denmark, http://www.
dako.com), and stained by 1:50 anti-Oct4 antibody (Santa
Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.
Cdx2 Regulates Bmp4 Expression in TS Cells
116
com). Anti-mouse Alexa 488 antibody was used as the second
antibody.
RESULTS
50 -Flanking Region of Mouse Bmp4 Has an
FGF4-Responsive Enhancer Element
To investigate the induction of Bmp4 expression in response
to FGF4 in TS cells, we analyzed the transcriptional activity
of the Bmp4 gene. The mouse Bmp4 gene has three noncoding exons designated Exon 1A, Exon 1B, and Exon 2 and two
coding exons designated Exon 3 and Exon 4 [27] (Fig. 1A).
Transcription of Bmp4 involves Exon 1A, 2, 3, and 4; Exon
1B, 2, 3, and 4; or Exon 2-4 [27, 33]. We constructed vectors
with intron 1A, intron 1B, a 1.4- or 2.3-kb fragment of the 50 flanking region of the Bmp4 gene in the luciferase cassette
vector. TS cells transfected with constructed vectors were cultured with or without FGF4 for 24 hours. We found that the
1.4-kb fragment taken from upstream of the 50 -flanking region
significantly increased the luciferase activity when it was
stimulated by FGF4 (Fig. 1A). This result suggests that the
1.4-kb fragment includes the FGF4-responsive enhancer element. To identify the enhancer element, we next made constructions of 200-bp deletions in the 1.4-kb fragment. Luciferase assays revealed that the second deletion (2118 to 1928
bp) and fifth to seventh deletion (1516 to 949 bp) caused
a significant reduction in luciferase activity (Fig. 1B). Then
we focused on this second deletion and made constructs containing 50-bp deletions (supporting information Fig. 1). The
first deletion (2118 to 2073 bp) resulted in a strong reduction of the transcriptional response to FGF4. Finally, we
made 12-bp deletion constructs and found that the third deletion (2096 to 2085 bp) had reduced activity in comparison
to the other deletion constructs (Fig. 1C). We found that the
third deletion contains the Cdx2-binding consensus sequence
‘‘TTTAT’’ [29]. These results raise a possibility that Cdx2 is
one of the regulatory factors of Bmp4 transcription.
Cdx2 Binds to the Consensus Binding Site in the
Mouse Bmp4 50 -Flanking Region
Next we performed EMSA to analyze whether Cdx2 binds to
the consensus sequence in the 50 -flanking region of Bmp4
(Fig. 1D). We found that the amount of DNA/protein complex
was larger in the nuclear extract of the FGF4-treated TS cells
than that of FGF4-untreated TS cells with DNA probe containing the Cdx2-binding site (Bmp4-wt) (black arrowhead,
lanes 2 and 8). A super shift complex was observed by addition of anti-Cdx2 antibody (white arrowhead, lane 3). When
we added unlabeled Bmp4-wt probe, the amount of the DNA/
protein complex was reduced (black arrowhead, lane 4). However, it was not reduced by addition of the Bmp4-mut probe,
which has mutation at the Cdx2-binding site of Bmp4-wt
(black arrowhead, lane 5). It is known that a promoter region
of MUC2 binds to Cdx2 through the consensus sequence [29].
The amount of the DNA/protein complex was reduced when
we added a DNA probe containing the Cdx2-binding site of
MUC2 (MUC-WT) [29] (black arrowhead, lane 6). However,
it was not reduced by the MUC-Mut probe carrying a mutation in the Cdx2-binding site of MUC-WT (black arrowhead,
lane 7). To confirm that Cdx2 binds to the Bmp4 50 -flanking
region containing the Cdx2-binding consensus sequence in TS
cells in response to FGF4, we performed ChIP assay. We
found that endogenous Cdx2 binding to the Bmp4 promoter
was increased when cells were stimulated with FGF4 (Fig.
Figure 2. Knockdown of Cdx2 by siRNA transfection. Trophoblast
stem cells were transfected with siRNA [siCdx2 or scramble oligonucleotides (as a control)] and, after 36 hours of culture, were harvested
for lysate or RNA isolation. Cell lysates were subjected to Western
blotting with anti-Cdx2 and anti-actin antibodies and RNA was subjected to quantitative reverse transcription-polymerase chain reaction.
The results were obtained from at least three separate transfections
and are indicated as the mean 6 SD. Abbreviations: BMP, bone morphogenetic protein; FGF, fibroblast growth factor.
1E). Thus, it appears that Cdx2 binds to the Cdx2 consensus
binding site ‘‘TTTAT’’ in the 50 -flanking region of Bmp4 in
TS cells in an FGF4-dependent manner.
Gene Silencing of Cdx2 Downregulates Bmp4
mRNA Expression
Because our findings suggest that Cdx2 activates transcription
of Bmp4 in an FGF4-dependent manner, we analyzed the time
course of the mRNA expression of Cdx2 and Bmp4 in TS
cells upon stimulation with or withdrawal of FGF4 by quantitative polymerase chain reaction (PCR) (Results in the supporting information and supporting information Fig. 2).
To analyze whether Cdx2 regulates Bmp4 expression in
cells, we knocked down Cdx2 in TS cells by siRNA (Fig. 2). We
analyzed expression levels of Cdx2 protein by Western blotting.
Expression levels of Cdx2 were greatly decreased by transfection
of siRNA for Cdx2. In the presence of FGF4, expression of
Bmp4 mRNA was downregulated up to half in Cdx2 knocked
down cells compared to that of control siRNA-transfected or
nontreated cells. These results indicate that Cdx2 is a major transcription factor that regulates expression of Bmp4.
Inhibition of the MEK-ERK Pathway
Downregulates FGF4-Induced Cdx2 Expression
and Bmp4 Secretion
To investigate which signal transduction pathways are responsible for the expression of Cdx2 and secretion of Bmp4, we
treated TS cells with an inhibitor specific to each pathway—
U0126, an inhibitor of MEK, SB202190, an inhibitor of p38
mitogen activated-protein kinase (MAPK), and LY294002, an
inhibitor of PI-3 kinase—and examined the expression levels
of ERK1/2, phospho-ERK1/2, Akt, phospho-Akt, Cdx2, and
Bmp4 by Western blotting (Fig. 3A). After 15 minutes of
stimulation with FGF4, ERK1/2 became phosphorylated and
the phosphorylation was inhibited by U0126 but not by
SB202190 or LY294002 (Fig. 3A). Phosphorylation of Akt
was not induced by FGF4 stimulation and it was downregulated by LY294002. After 24 hours of stimulation with FGF4,
the expression of Cdx2 and secretion of Bmp4 were observed;
Murohashi, Nakamura, Tanaka et al.
117
Inhibition of the MEK-ERK Pathway Downregulates
the FGF4-Responsive Enhancer Activity of the
50 -Flanking Region of Bmp4
To examine whether the FGF4-responsive enhancer activity is
also dependent on the MEK-ERK pathway, we performed luciferase assay with the 50 -1.4 kb vector after treatment with
the inhibitors. Treatment with U0126 but not with SB202190
or LY294002 caused a reduction of the activity, revealing
that the enhancer activity of the Bmp4 gene is dependent on
the MEK-ERK pathway (Fig. 3B).
We then examined whether there exist PI-3 kinase-regulatory elements in the 50 -flanking region of the Bmp4 gene. We
analyzed luciferase activity with the constructs used in Figure
1A with or without LY294002 treatment in the presence or
absence of FGF4 (data not shown). However, no constructs
showed significant difference in luciferase activity with the
treatment with LY294002. It is thus unlikely that these
regions contain such PI-3 kinase-regulatory elements.
Exogenous Bmp4 Rescues the Defective
Growth of ICM-Derived Cell Mass in
Cultured Frs2a-Null Embryos
Figure 3. Effects of inhibitors on the FGF4 signaling pathway. (A):
Immunoblotting of the TS cell lysate or culture supernatant. TS cells
were starved for 6 hours, treated with 10 lM of the inhibitor U0126,
SB202190, or LY294002 or with DMSO (control) for 1 hour and
stimulated with FGF4 (50 ng/ml) and Heparin (5 lg/ml) for 15
minutes or 24 hours. Cell lysates were prepared from the 15-minuteor 24-hour-stimulated TS cells. Culture supernatants were prepared
from the 24-hour-stimulated TS cells. Quantitated data of each protein
were divided by the value of samples treated with FGF4 and DMSO.
We repeated experiments at least three times and obtained similar
results. (B): Effects of inhibitors on transcriptional activity of the
enhancer element of the Bmp4 promoter. TS cells were transfected
with the 50 -1.4 kb vector, treated with 10 lM inhibitor U0126,
SB202190, or LY294002 or with DMSO (control) for 1 hour and
stimulated with or without FGF4 (50 ng/ml) and Heparin (5 lg/ml)
for 24 hours. Cell lysates were prepared and transcriptional activity
was measured. The results were obtained from at least three separate
transfections and are indicated as the mean 6 SD. Abbreviations:
BMP, bone morphogenetic protein; DMSO, dimethyl sulfoxide; ERK,
extrocellular signal-regulated protein kinase; FGF, fibroblast growth
factor; TS, trophoblast stem.
however, both were inhibited by treatment with U0126 but
not with SB202190 or LY294002. From these results, we concluded that the MEK-ERK pathway but not p38 MAPK pathway or PI-3 kinase pathway is responsible for both expression
of Cdx2 in TS cells and secretion of Bmp4 from TS cells.
Interestingly, the secretion of Bmp4 was increased when
Akt phosphorylation was reduced by LY294002 treatment (Fig.
3A). Because the PI-3 kinase/Akt pathway is known to mediate
the cell survival pathway and transcription factors FoxO family
are negatively regulated by the PI-3 kinase/Akt pathway [35,
36], we examined whether the increase of Bmp4 secretion by
treatment with LY294002 was related to cell death by fluorescence-activated cell sorting analysis (Results in the supporting
information and supporting information Fig. 3) and mRNA
expression of Fox families in TS cells (Results in the supporting
information and supporting information Fig. 4). These results
suggest that the increase of Bmp4 protein is not related to cell
death and that the PI-3 kinase/Akt pathway does not regulate
Fox family members at mRNA levels in TS cells.
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We have previously shown that Frs2a/ embryos are small and
have reduced levels of ERK activation and Bmp4 expression in
the ExE in association with a reduction in the amount of phosphorylated Smad1/5 in the epiblast [15]. Furthermore, Frs2a/ blastocysts from which TS cell lines could not be established had
reduced levels of expression of Cdx2. We cultured Frs2a/ blastocysts and observed outgrowth of the blastocysts for 4 days. The
lower layer is an adherent sheet of TE-like cells including trophoblastic giant cells and the ICM-derived cells that laid on top of the
lower layer [5]. We stained cultured Frs2/ mutant and wildtype or heterozygous embryos (nonmutant) blastocysts with alkaline phosphatase (ALP) after 4-day culture. We also examined
expression of Oct4, a marker for the ICM. The ICM-derived cell
mass was positive for ALP and Oct4 in nonmutant and Frs2a/
embryos (Fig. 4A–4H).
We found that the Oct4-positive ICM-derived cell mass
was smaller in Frs2a/ embryos compared with that of nonmutant embryos. To quantify this result, we first measured the
diameter of the adherent sheet of TE-like cells and found that
there was no significant difference in the diameter between
nonmutant and mutant embryos. (The ratio, mutant/nonmutant, was 0.94 6 0.13 [p ¼ .417]). Then we measured the diameter of the ICM-derived cell mass and calculated the ratio
(ICM/TE). Because the diameter of the adherent sheet of TElike cells showed no significant difference as described above,
the ratio of the diameters of these two cell populations, ICM/
TE, should reflect the growth of ICM-derived cell mass at
some extent. As a result, the ratios were smaller for mutant
embryos than for nonmutant embryos, suggesting that the
growth of ICM-derived cells were defective in mutant
embryos (Fig. 4I, 4K, 4M; black columns).
To examine whether the phenotype of the mutant is
caused by decreased secretion of Bmp4 from the TE, we
added 100 ng/ml recombinant Bmp4 or 0.1% bovine serum
albumin (as a control, the same volume as Bmp4 solution) to
each well after 1 and 3 days. There was no significant difference in the diameter of the adherent sheet of TE-like cells in
the presence or absence of Bmp4 in either nonmutant or mutant embryos. [The ratio, Bmp4-treatment/control, was 1.06 6
0.17 (p ¼ .331) for nonmutant and 0.946 6 0.10 (p ¼ .327)
for mutant]. The ICM-derived cell mass remained as Oct4positive in the presence of Bmp4 (data not shown). No difference in the ICM/TE ratio was observed in nonmutant blastocysts with or without Bmp4 (Fig. 4I, 4J, 4M; left columns).
118
Cdx2 Regulates Bmp4 Expression in TS Cells
Figure 4. Analysis of cultured
blastocysts in vitro. E 3.5 blastocysts derived from Frs2aþ/ intercrosses were cultured with TS
medium and 0.1% BSA (black columns) or 100 ng/ml Bmp4 (white
columns) for 4 days. (A–D): Blastocysts were cultured for 4 days
and stained by alkaline phosphatase or anti-Oct4 antibody for
detecting ICM-derived cells (A, C,
E, G) and Hoechst 33342 (B, D,
F, H). Mutant blastocysts (C, D,
G, H) had a smaller ICM compared with nonmutant blastocysts
(A, B). The ICM of mutant blastocysts treated with Bmp4 (K,L)
grew to the same level as that of
nonmutant blastocysts (I, J).
Arrows indicate the ICM. |: A long
axis for measuring the diameter of
ICM. Scale bars: 100 lm. White
dashed circle indicates the outline
of the TE and black circle indicates the outline of the ICM (I).
The longest diameters (straight
lines) were measured. (M): The ratio of diameters of ICM to TE
(ICM/TE) of 4-day-culture nonmutant (wild-type and heterozygote)
and mutant blastocysts (n ¼ 11 for
wild type, n ¼ 22 for heterozygote,
and n ¼ 10 for mutant). p ¼ .0242
(left) and p ¼ .0378 (right). The
results are indicated as the mean 6
SD. (N): The ratio of areas of ICM
to TE (ICM/TE) of 4-day-culture
wild-type blastocysts treated with
0.1% BSA (n ¼ 8) or 500 ng/ml
Noggin (n ¼ 11), p ¼ .0214. The
results are indicated as the mean 6
SD. Abbreviations: ALP, alkaline
phosphatase; BMP, bone morphogenetic protein; BSA, bovine serum albumin; FGF, fibroblast
growth factor; ICM, inner cell
mass; TE, trophectoderm; TS,
trophoblast stem.
In contrast, there was a significant increase in the ICM/TE ratio when mutant embryos were treated with exogenous Bmp4
(Fig. 4K–4M; right columns). These results suggest that exogenous Bmp4 can rescue the defective growth of ICM-derived
cell mass in the Frs2a/ embryos and raise a possibility that
the FRS2a-Bmp4 pathway plays an important role for proper
growth of the ICM and epiblast in vivo.
remained as Oct4-positive in the presence of Noggin (data not
shown). The average area ratio of the ICM to that of the TE was
smaller for Noggin-treated embryos, although there was no significant difference in the area of the adherent sheet of TE-like cells in
the presence or absence of Noggin. [The ratio, Noggin-treatment/
control, was 1.16 6 0.16 (p ¼ .154)] (Fig. 4N). This result suggests that Bmp4 supports growth of the ICM-derived cell mass.
Treatment with Noggin Reduces Growth of the
ICM-Derived Cell Mass of Nonmutant Embryos
Possible Involvement of Other Transcription Factors
for Bmp4 Expression in Response to FGF4
To further examine the effects of Bmp signal on growth of the
ICM and TE, we added Noggin, a Bmp antagonist, in the culture
medium of wild-type blastocysts obtained from crossing of Swiss
Webster and BL6/J background mice. The ICM-derived cell mass
Transcription of Bmp4 with stimulation of FGF4 may be
regulated not only by Cdx2 but also by other transcription
factors. Indeed, we found that a relatively large region comprised of three parts of the 50 -flanking region of Bmp4, from
Murohashi, Nakamura, Tanaka et al.
1516 to 1297 bp, 1296 to 1104 bp, and 1103 to
949 bp, contributes to an increase in the transcriptional activity of Bmp4 in response to FGF4 (Fig. 1B). By sequence
alignment we found that there are multiple potential binding
sites for c-Ets family proteins or AP-1, early response transcription factors. To examine the contribution of AP-1 and cEts in an early phase of FGF4 response, we analyzed the
expression of Ets1 and c-Fos, a component of AP-1, with or
without FGF4 stimulation within 60 minutes (supporting
information Fig. 5). Interestingly, although Ets1 did not show
significant difference by FGF4 stimulation, the expression of
c-Fos was significantly increased within 60 minutes. These
results raise a possibility that transcription of Bmp4 is regulated by AP-1 in TS cells in response to FGF4.
DISCUSSION
In this paper, we describe novel molecular mechanisms for
regulating the stem cell niche. A major player is found to be
a transcription factor Cdx2, under the control of which no
genes have yet been identified in the TE or TS cells.
In the present study, we clearly demonstrated that Cdx2 is
one of the key transcription factors for activation of the transcription of Bmp4 in TS cells. In addition, we showed a possibility that AP-1 also regulates the transcription of Bmp4 in
TS cells. It is known that c-Ets and AP-1 regulate the transcription of a variety of genes and are activated downstream
of ERK [37]. Moreover, a previous report suggests that AP-1
activates Bmp4 transcription in Xenopus embryos [28]. Considering that c-Ets and AP-1 are expressed more broadly than
Cdx2, we propose that Cdx2 is a cell type-specific transcription factor for activation of the transcription of Bmp4 and that
Cdx2 and other transcription factors such as AP-1 cooperatively activate transcription of Bmp4 to be expressed efficiently in response to FGF4 (see discussion below). It would
be interesting to examine if expression of Bmp4 is induced in
response to FGF4 in other cell types such as intestinal epithelial cells where Cdx2 is known to be expressed.
Another interesting finding of the luciferase assays is that
the 50 -2.3 kb fragment of the 50 -flanking region of Bmp4
showed reduced activity in response to FGF4, as compared
with the 50 -1.4 kb fragment. This raises the possibility of
some suppressor elements between 949 and 1 bp. We are
making constructs that have deletions in this region and are
carrying out luciferase assays. The results will be presented in
a future paper.
We analyzed the time course of the expression of Cdx2
and Bmp4 by using a quantitative RT-PCR system to obtain
insight into how the expression of each gene is regulated.
There are two waves of increases in Cdx2 mRNA but the
mechanisms are unknown. Because transcription is regulated
by positive and negative regulatory factors, it is possible that
positive factors such as AP-1 rather than negative factors are
preferentially induced in the early phase of FGF4 stimulation.
Then negative factors are induced and cooperative effects of
both positive and negative factors would emerge in the late
phase. However, there was no first wave response of Bmp4
mRNA. Because expression of AP-1 is also dependent on
FGF4 signaling, it might take time for them to accumulate in
cells to efficiently activate transcription of Bmp4. In addition,
we found that expression levels of Cdx2 showed an increase
for 2 hours by FGF4 withdrawal. It could be that negative
regulatory factors bind to suppressor elements of Cdx2 and
that, upon withdrawal of FGF4, they are apt to be released
from the DNA or degraded faster than positive regulators.
www.StemCells.com
119
We showed that protein levels of Cdx2 and secretion levels of Bmp4 were dependent on the MEK-ERK pathway in
TS cells. Furthermore, the enhancer activity of Bmp4 was
also dependent on the MEK-ERK pathway. Therefore, it
appears that FGF4 stimulates the MEK-ERK pathway and
induces the expression of Cdx2, and then Cdx2 activates the
transcription of Bmp4, leading to an increase in Bmp4 secretion. We found that activation of Akt did not occur in TS
cells in response to FGF4. Although FRS2a is abundantly
expressed in TS cells [15] and it activates not only ERK but
also PI-3 kinase in some cells [16], it appears that FRS2a
mainly contributes to the activation of ERK in TS cells. There
seems to be cell-context-dependent mechanisms for activation
of PI-3 kinase in response to FGF4.
In addition, it appears that production of Bmp4 is negatively regulated by Akt. We also found that PI-3 kinase inhibits expression of Bmp4 at the transcription levels. The inhibitory effects of PI-3 kinase emerge when the FGF4 signaling
pathway is activated because PI-3 kinase inhibitor did not
induce expression of Bmp4 in the absence of FGF4. Thus,
FGF4 signaling is required for expression of Bmp4 and the
PI-3 kinase/Akt pathway appears to fine-tune the expression
levels of Bmp4, although molecular mechanisms are not yet
clarified. ERK activation, expression of Cdx2 or Fox family
transcription factors, is unlikely to be a direct target of the inhibition of the PI-3 kinase pathway. It has been reported that
phosphorylation of FoxO family proteins by Akt inhibits its
transcription activity [35, 36]. It is tempting to speculate such
mechanisms might operate for regulation of Bmp4 expression
in TS cells. Although the 50 -flanking region of Bmp4 gene,
which was investigated in this paper, does not appear to have
regulatory elements by PI-3 kinase, it is reasonable to believe
in the possibility that there exist such elements in other
genomic regions.
A knockdown analysis provided strong evidence that
Cdx2 regulates expression of Bmp4. Although a great reduction of Cdx2 was achieved by siRNA, the downregulation of
Bmp4 was not complete. The expression of remaining Bmp4
could be explained by the effects of other transcription factors
that are downstream from FGF4 signaling such as AP-1 or cEts. Thus, the transcription of Bmp4 can be cooperatively
regulated by Cdx2 and other transcription factors. In addition,
it is equally possible that knockdown of Cdx2 was not complete and that Bmp4 expression still remained.
By culturing blastocysts, we found that Frs2a-null embryos
could attach to the dishes and expand, but that they showed
growth retardation in the ICM. In contrast, growth of the TE
layer appeared to show no difference between the mutant and
wild-type or heterozygote after 4 days. Because the primary
defect of Frs2a mutant embryos is in the maintenance of TS
cells in the TE and ExE [15], we first hypothesized that the phenotype might appear in the TE. However, the result was not as
we predicted. We have previously shown that TS cell colonies
appeared derived from cultured Frs2a-null blastocysts but that
they disappeared after several passages. This indicates that the
Frs2a mutant embryos have some TS cells at the blastocyst
stage [15]. Thus, the TE layer in 4-day-cultured embryos may
reflect only the initial number of TS cells. Although the phenotype of the TE may appear after a longer culture, it is impossible to check this because of loss of viability of the embryos.
Moreover, it is reported that Fgf4-null or Fgfr2-null blastocysts
also had little or no ICM with the similar assay [3, 5]. The striking similarity in the phenotypes of Frs2a-null, Fgf4-null, and
Fgfr2-null reveals that the FGF4-FGFR2-FRS2a pathway may
play a critical role in the growth of the ICM.
Our results suggest that FRS2a-mediated production of
Bmp4 in TS cells is important for the growth of the ICM-
Cdx2 Regulates Bmp4 Expression in TS Cells
120
derived cells. Indeed, Bmp4 was necessarily identified as a
factor in a conditioned medium of mouse embryonic fibroblasts to maintain the pluripotency of ES cells in the presence
of leukemia inhibitory factor in vitro [38]. Moreover, analysis
of Bmp4-null embryos revealed that Bmp4 may be required
for epiblast cell proliferation [39]. In addition, this study also
shows that Bmp4 is not genetically required for trophoblast
formation because development of trophoblast is apparently
normal in Bmp4-null embryos. All of these findings support
the notion that Bmp4 promotes growth of the ICM-derived
cells.
The transcription factor Cdx2 is important for the establishment and maintenance of intestinal epithelial cells and
well known as a marker of colon cancer. It regulates the transcription of several genes like MUC2 expressed in mucin-producing secretory goblet cells in the intestine [29]. In this
study, we found the role of Cdx2 in TS cells: induction of the
expression of Bmp4, the first gene identified as a target of
Cdx2 in TS cells.
It was recently reported that the FGF4-Shp2-ERK signaling pathway controls TS cell survival through downregulation
of the expression of the proapoptotic protein Bim based on a
study of Shp2-null embryos [40]. Given that the activation of
Shp2 is largely mediated by FRS2a, the FGF4-FRS2a-Shp2ERK pathway appears to bifurcate at the level of ERK. One
route is for the survival of TS cells and the other is for
expression of Cdx2 mRNA. Expression of Cdx2 is stimulated
by the activation of ERK at the transcription level, at least
partly through Cdx2-promoter binding to Ets2, a transcription
factor [12, 41].
Taken together, we propose that FGF4 and Bmp4 provide
a paracrine mechanism in early mammalian embryos where
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from FGF4-FGFR2-FRS2a-ERK-Cdx2 to Bmp4 in TE leads
to Bmp4 secretion. Then, Bmp4 appears to play an important
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ACKNOWLEDGMENTS
We thank Dr. T. Kurihara for providing the mouse Bmp4
genomic fragment cloning vector. We are grateful of Drs. H. Iba
and T. Tango for helping to perform ChIP experiments. We
thank Drs. H. Koide and T. Yokota for critical reading of the
manuscript. This work was supported by Grants-in-Aid for Special Project Research on Stem Cells (17045008) to N.G. and on
Cancer-Bioscience (17013025) to N.G. and (17014020) to M.S.
from the Ministry of Education, Science, Sports and Culture of
Japan and by NOVARTIS Foundation (Japan) for the Promotion
of Science to N.G. J.S. was supported by NIH grants AR 051448
and AR 051886. T.N. is currently affiliated with the Department
of Genetics and Complex Diseases, School of Public Health,
Harvard University, Boston, MA.
DISCLOSURE
OF
OF
POTENTIAL CONFLICTS
INTEREST
The authors indicate no potential conflicts of interest.
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