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J Biol Chem, Vol. 274, Issue 28, 19838-19845, July 9, 1999
From the Bone morphogenetic proteins
(BMPs) are morphogenetic signaling molecules
essential for embryonic patterning. To obtain molecular insight into
the influence of BMPs on morphogenesis, we searched for new genes
directly activated by BMP signaling. In vitro cultured mouse embryonic stem (ES) cells were used, cultivated in chemically defined growth medium (CDM). CDM-cultured ES cells responded very selectively to stimulation by various mesoderm inducers (BMP2/4, activin A, and basic fibroblast growth factor). BMP2/4 rapidly induced
transcript levels of the homeobox genes Msx-1 and
Msx-2 and the proto-oncogene JunB, whereas
c-jun transcripts displayed delayed albeit prolonged
increase. Using differential display cDNA cloning, six direct BMP
target genes were identified. These include Id3, which
showed strong mRNA induction, and the moderately induced
Cyr61, DEK, and eIF4AII genes, as
well as a gene encoding a GC-binding protein. Besides Id3,
also the Id1 and Id2 genes were activated by
BMP4 in both ES cells and a range of different cell lines.
Id genes encode negative regulators of basic
helix-loop-helix transcription factors. In vivo we observed
local ectopic expression of Id3 and Msx-2
mRNAs in Ft/+ embryos at overlapping regions of ectopic
Bmp4 misexpression. We therefore propose that the
Msx and Id genes are direct target genes of
embryonic BMP4 signaling in vivo.
One important stage in the development of the vertebrate embryo is
reached when the three germ layers are formed and the body plan gets
established during the process of gastrulation (1, 2). Our
understanding of the molecular mechanism mediating gastrulation is
still insufficient, although over the past years many secreted growth
factor-like molecules have been identified to play important roles in
these early embryonic events (3). Among these signaling molecules are
several members of the BMP1
family. BMP-related growth factors belong to the TGF- Cells--
Cells used in this study were CCE (29, 32), E14.1
(33), C3H/10/T1/2 (ATCC, CCL-226), C2C12 (ATCC, CRL-1772), PC-12 (ATCC, CRL-1721), Saos-2 (ATCC, HTB-85), U-2 OS (ATCC, HTB-96), 3T3-L1 (ATCC,
CCL-92.1), Raji (ATCC, CCL-86), MOLT-4 (ATCC, CRL-1582), HeLa (ATCC,
CCL-2), HL-60 (ATCC, CCL-240), and RK13 (ATCC, CCL-37).
ES Cell Culture and Stimulation Conditions--
The used ES cell
line CCE (129/Sv/Ev-derived) was already adapted to grow in the absence
of feeder cells. The cells were routinely grown on gelatinized flasks
in Dulbecco's modified Eagle's medium supplemented with 1000 units/ml
leukemia inhibitory factor, 15% heat-inactivated fetal calf serum, 150 µM
For stimulation studies, ES cells were trypsinized, washed free of
leukemia inhibitory factor and FCS, and seeded in 10 ml of CDM medium
at a density of 5 × 105 cells per 9-cm diameter
gelatinized tissue culture dish. CDM was prepared as described (29).
The medium was changed every 2nd day. If not stated otherwise, cells
were stimulated on day 5 of cultivation for the indicated times using
BMP2 (5 ng/ml), BMP4 (5 ng/ml), activin A (20 ng/ml), bFGF (20 ng/ml),
FCS (10%), or 12-O-tetradecanoylphorbol-13-acetate (1 µM). Cycloheximide (100 µg/ml) treatment started 30 min
and actinomycin D (5 µg/ml) treatment 15 min prior to the addition of
the inducers. We used CHX at concentrations that are known to inhibit
completely cellular protein synthesis of embryonal cells (35, 36).
C3H/10T1/2, 3T3-L1, Saos-2, and U-2 OS cells were routinely cultured in
tissue culture flasks in Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated FCS, 0.2 mM
L-glutamine, and antibiotics (50 units/ml penicillin, 50 µg/ml streptomycin); for C2C12 cells the concentration of FCS was
increased to 20%. PC-12 cells were maintained in RPMI medium (16:40)
supplemented with 10% heat-inactivated horse serum, 5%
heat-inactivated FCS, 0.2 mM glutamine, and antibiotics (50 units/ml penicillin, 50 µg/ml streptomycin). For stimulation studies
cells were plated at a density of 1 × 106 cells per
9-cm dish and were cultured for 30 h in the appropriate culture
medium, as outlined above. Then the cells were washed three times with
PBS, followed by an additional 48 h incubation in CDM.
Stimulations with the indicated effector molecules were done as
described above for the ES cells.
RNA Isolation and Northern Blot Analysis--
Total RNA was
prepared from cell lines using the UltraspecTM RNA
isolation system (Biotecx Laboratories, Inc.). Samples of 10 µg of
RNA were separated electrophoretically on 0.8% agarose, 2.2 M formaldehyde, 1× RNA borate buffer gels, stained with
ethidium bromide to monitor equivalence of loading by fluorescence
intensity of rRNA bands, and blotted onto Genescreen plus nylon
membranes (NEN Life Science Products). Prehybridization and
hybridization were carried out in Church Buffer (250 mM
Na2HPO4, pH 7, 7% SDS) at 65 °C. Probes
were labeled using a random prime labeling kit (Prime-itTM
II, Stratagene) with [ Differential Display (dd) RT-PCR--
The RNA
ImageTM Kit protocol (GenHunter Corp., Nashville, TN) was
largely followed. Total RNA from CCE cells, mock-treated with either
vehicle (1% bovine serum albumin in PBS) or treated with BMP-2 (or
BMP-4) in the presence of cycloheximide, was purified by guanidinium
thiocyanate/cesium chloride step gradients (38). After treatment with
RNase-free DNase to remove any DNA contamination, aliquots of each RNA
were used as templates for three different first strand cDNA
synthesis reactions using three different one-base anchored oligo(dT)
primers, respectively (T11G, T11A, and
T11C). Aliquots of these cDNAs served as templates for
subsequent PCR amplifications using a combination of an arbitrary
13-mer oligonucleotide as 5'-primer and a 3'-primer identical to the
one-base anchored T11 oligonucleotide used in the cDNA
synthesis reaction. All primers used contained additional
HindIII restriction sites (5'-AAGCTT-3') at the 5'-ends to
facilitate manipulation of the amplified cDNAs after cloning. PCR
was performed as follows: 94 °C, 30 s; 40 °C, 2 min;
72 °C, 30 s; for 40 cycles, with a final extension phase at
72 °C for 5 min. The PCR fragments were labeled by amplification in
the presence of [ Cloning and Sequencing--
Reamplified cDNA fragments were
cloned into the pGEMR T-vector using the TA cloning system
(Promega). Cloned cDNAs were sequenced with the
CyclistTM Taq DNA Sequencing Kit (Stratagene)
using either T7 or Sp6 primers. DNA data bank searches were performed
using the "NCBI nr" data base.
Western Blotting--
Whole cell extracts were prepared in
Fracelton buffer as described (39). Cell lysates (20 µg/lane) were
separated by SDS-15% polyacrylamide gel electrophoresis prior to
electrophoretic transfer onto nitrocellulose (Optitran BA-S 85, Schleicher & Schuell, Germany) or polyvinylidene fluoride microporous
membranes (Immobilon-P, Millipore Corp., Bedford, MA). The transfer was
done under semi-dry conditions (40) in transfer buffer (50 mM Tris, 40 mM glycine, 375 mg/ml SDS, 20% v/v
MeOH) for 2 h at 0.8 mA/cm2. Blots were blocked with
5% (w/v) nonfat dry milk in TBST (20 mM Tris-HCl, pH 8.0, 0.9% NaCl, 0.05% v/v Tween 20) for 1 h and were then incubated
with one of the following primary polyclonal rabbit antisera (Santa
Cruz Biotechnology): Id1 (C20, catalog number sc-488), Id2 (C20,
catalog number sc-489), and Id3 (C20, catalog number sc-490). The
antibodies were diluted 1:1000 in 5% (w/v) nonfat dry milk in TBST,
and incubations were performed for 12 h. Blots were then washed
three times for 5 min in TBST and subsequently incubated for 1 h
with the secondary anti-rabbit horseradish peroxidase-conjugated
antibody at 1:2500 dilution (using 5% (w/v) nonfat dry milk in TBST).
Signals were visualized with the ECL system (Amersham Pharmacia
Biotech) and were sized relative to the positions of a prestained size
marker (Life Technologies, Inc.).
Animals--
The dominant mouse mutant Fused toes (Ft) was
generated by transgene integration into F2(C57BL6xSJL)
embryos (41). Ft/+ mutants were subsequently back-crossed to C57BL6
over more than 20 generations. By crossing Ft/+ mice to NMRI mice the
line Ft(NMRI) was generated. The F1 generation of these
Ft(NMRI) crosses was used in all experiments. Embryos were genotyped by
PCR as described previously (42). Developmental stages were assigned
according to Hogan et al. (43).
Whole Mount in Situ Hybridization--
The in situ
hybridizations and control hybridizations were done according to Heymer
et al. (42) with the following additional steps. Subsequent
to fixation of the embryos in 4% paraformaldehyde, they were processed
first through 50% methanol for 15-30 min and then to 100% methanol
for at least overnight at BMPs and bFGF Elicit Specific and Distinct Transcriptional
Responses in ES Cells Cultured in Chemically Defined Growth
Media--
To study patterns of gene expression induced in embryonic
stem cells by stimulation with mesoderm inducing factors (BMP2, BMP4,
activin A, and bFGF), ES cells (CCE) were cultivated as monolayers on
gelatinized plates in the absence of feeder cells. To prevent undesired
effects exerted by ill-defined components present in serum-containing
growth media, we cultivated the ES cells in chemically defined medium
(CDM) (29). Such cultured cells responded very sensitively and
specifically to the application of different individual stimuli, as
judged by transcript levels of the marker genes Msx-1,
Msx-2, c-fos, and Egr-1 (Fig.
1A). Application of BMP4 led
to a rapid and lasting induction of the homeobox genes Msx-1
and Msx-2, while hardly affecting c-fos and Egr-1 expression (Fig. 1A, compare lanes
1-5 with lane 11). An identical response was elicited
upon induction with BMP2 (data not shown). Activin A treatment led to a
transcriptional response very similar to the one induced by BMPs,
except that Msx gene induction was somewhat weaker and less
long-lived (not shown). In contrast, treatment with bFGF rapidly
induced c-fos and Egr-1 transcripts while having
only marginal effects on expression of Msx-1 and
Msx-2 (Fig. 1A, lanes 6-11). None of these
stimuli affected expression of the genes encoding T (Brachyury) or
Goosecoid (not shown) during the time course shown, except that activin
A induced the T gene after the 1st day of treatment (not
shown). Identical results were obtained with the ES cell line E14.1
(data not shown).
Since members of the Jun family of transcription factors, including
c-Jun, JunB, and JunD, play important roles in programs of cell growth
and differentiation (44, 45), we measured the ES cell responses of
these genes to BMP4 treatment. c-jun was induced in a
delayed fashion by BMP4, with transcripts still accumulating up to
24 h (Fig. 1B, lanes 1-16). In contrast,
JunB expression was induced only weakly and transiently
(Fig. 1B, lanes 17-21). Both these genes are rapidly
induced by the inhibitor of protein synthesis cycloheximide (CHX), a
response that is enhanced in the presence of BMP4 in the case of
JunB but not in the case of c-jun (Fig. 1B,
lanes 22-25). The JunD message was not affected under
any circumstance (data not shown).
This analysis demonstrates, in agreement with Johansson and Wiles (29),
that ES cells, when cultured in chemically defined medium, are able to
activate distinct gene expression programs in response to specific
extracellular stimuli.
The Msx-1 and Msx-2 Genes Exhibit Immediate Early-type Responses to
BMP4 in ES Cells--
Previous studies demonstrated that BMP4 can lead
to up-regulation of Msx gene expression in distinct
embryonic domains, including dental mesenchyme (46), hindbrain (47),
neural tube (48), limb bud (49), paraxial ectomesoderm, and facial
tissues (50). It was unclear whether the Msx genes were
directly activated by BMPs or indirectly. Therefore, we used our ES
cell system to examine whether induction of the Msx-1 and
Msx-2 genes represented a direct and possibly immediate
early-type response to BMP4. CCE cells were incubated for 2 h with
BMP4 in the presence or absence of cycloheximide.
In Fig. 2 we first show that induction by
BMP4 of both Msx-1 and Msx-2 transcripts
represents an extended response, lasting for 24 h (Fig. 2A,
lanes 1-16) and even up to 3 days (not shown). The induction
measured after 2 h of BMP4 treatment is unaffected in the presence
of cycloheximide (Fig. 2B, lanes 1-4). Since uninduced levels of the Msx genes are not detectable at this time
point (lane 1) and since CHX alone only leads to a marginal
signal possibly due to message stabilization (lane 2), the
revealed immediate early response of the Msx-1 and
Msx-2 genes toward BMP4 suggests that these genes are direct
target genes of BMP signaling. Identical results were obtained with the
ES cell line E14.1 (data not shown).
By using CCE cells grown in CDM in the presence of high concentrations
of BMP4 (>2 ng/ml) Johannson and Wiles (29) reported transient
expression of mesoderm markers and subsequently the formation of
hematopoietic precursor cells accompanied by the expression of
corresponding lineage markers. Additionally, we performed dose-response
experiments which showed that application of BMP2/4 at 0.5 ng/ml
already led to a moderate enhancement of Msx-1 and
Msx-2 transcripts, which increased upon using BMP
concentrations up to 5 ng/ml. These responses were not enhanced any
further at higher BMP concentrations (data not shown). On the basis of
these results, we subsequently used CCE cells treated with BMP 2/4 at 5 ng/ml, i.e. treatment conditions eliciting maximal responses regarding Msx induction.
Isolation and Identification of New BMP-regulated Transcripts from
ES Cells Using Differential Display RT-PCR--
By having demonstrated
that CDM-cultured ES cells respond efficiently and selectively to BMP
signaling, we used this cellular system to identify by DD-RT-PCR (31)
new transcripts whose expression is induced by BMP4. Specifically, we
searched for genes whose induction by BMP fulfilled the criteria of an
immediate early-type response, i.e. displaying rapid and
transient transcriptional induction in the absence of de
novo protein synthesis (51). We searched for dd-RT-PCR products
differentially displayed in BMP-stimulated versus
mock-treated cells. Both BMP2 and BMP4 treatments were done in the
presence of cycloheximide. The presence of cycloheximide ensured the
isolation of those transcripts synthesized in an immediate early-type response.
60 separate DD-RT-PCR products were scored positive on differential
display gels, excised, reamplified, cloned, and reinvestigated for
differential expression by Northern blotting (not shown). We derived 15 independent cDNA clones that were positive in detecting mRNAs
stimulated by BMP (both BMP2 and BMP4, respectively), CHX, or combined
BMP/CHX treatment. Nine of these mRNAs were induced by CHX and not
by BMP, and six were stimulated by BMP alone and also by a combined
BMP/CHX treatment. These latter six cDNA clones, corresponding to
BMP-induced "immediate early" transcripts, were characterized
further by sequencing. Five cDNAs could be identified by sequence
homology, whereas one (clone A79) represented a hitherto unidentified
gene (Fig. 3A). Clone A51
exhibits homology to the human DEK oncogene (52), and clone
A52 represents a part of the Cyr61 gene that was first
identified as a growth factor-inducible immediate early gene in mouse
fibroblasts (53). Clone A59 corresponds to a gene encoding the RNA
helicase translation initiation factor eIF4AII (54), and clone C74
harbors part of a gene encoding an Sp1-like GC-binding protein (55).
The transcripts for these five cDNAs were moderately induced upon
BMP treatment alone (1.6-2.2-fold), whereas the mRNA corresponding
to clone G9 showed a strong induction (Fig. 3). Interestingly, clone G9
was identified as part of the cDNA encoding the Id3 protein, which
is a negative regulator of basic helix-loop-helix transcription factors
(56). Upon incubation of the ES cells with BMP4 in the presence of the
RNA synthesis inhibitor actinomycin D, the mRNAs corresponding to
all of the six isolated clones were not generated (Fig. 3A).
This suggests that BMP-induced generation of these mRNAs is
mediated, at least in part, at the level of transcription.
Id1, Id2, and Id3 Exhibit Immediate Early Responses to BMP4 in ES
Cells--
Four members of the Id family of HLH proteins have been
identified so far (Id1 to Id4) (56). We were interested to see whether, in addition to the Id3 gene identified above, any of the
other Id genes also responded to BMP4 treatment in ES cells.
Indeed, Id1 and Id2 transcripts were also rapidly
raised upon BMP4 stimulation (Fig. 3B and Fig.
4A, lanes 1-16), whereas no
expression of Id4 was observed in our experiments. The
inductions of Id1, Id2, and Id3 were blocked by
cotreatment with actinomycin D (Fig. 3B). Cycloheximide
treatment alone enhanced mRNA levels of these genes (Fig. 4B,
lanes 1 and 2), and this effect contributed to the
further increased expression levels seen after cotreatment with BMP4
and CHX (Fig. 4B, lanes 3 and 4). Collectively,
this argues that BMP-mediated induction of the Id1, Id2, and
Id3 transcripts occurred at the level of transcription, in a
fashion independent of de novo protein synthesis.
Furthermore, these inductions result in increased Id protein levels
(Fig. 4C).
Id1, Id2, and Id3 mRNAs Are Up-regulated by BMP4 in Different
Cell Types--
During embryogenesis the expression patterns of
Id genes overlaps with the expression of Bmp2 and
Bmp4 on many sites, for example during development of bone,
cartilage, whiskers, teeth, and gut (57, 58). In addition, BMP2 has
been shown to induce Id1 in osteoblast-like and myoblastic
cells (59, 60) which strongly suggested the BMPs as candidates of
Id gene-inducing polypeptides. Based on the hypothesis that
Id genes may serve as direct targets for BMP signaling in
many different organs, we examined by Northern analysis a range of cell
types for Id gene inducibility upon BMP4 treatment. The
Id1, Id2, and Id3 genes were
BMP-inducible in murine mesenchymal C3H/10T1/2 cells, murine preadipocytic 3T3-L1 cells, murine myocytic
C2C12 cells, human osteogenic Saos-2 and U2-OS
cells, and rat adrenergic PC-12 cells (Fig.
5). The kinetics of induction and
magnitudes of response appeared highly variant between these different
types of cell. Additionally, in human T-lymphocytic MOLT-4 and
B-lymphoblastic Raji cells, the Id mRNA levels were
increased only very moderately and transiently by BMP4 treatment. In
human epithelial HeLa cells, promyelocytic HL-60 cells, and
fibroblastic rabbit kidney RK13 cells, BMP did not elicit
any change in Id transcripts at all (data not shown).
Ectopic BMP4 Misexpression in Vivo Correlates with Up-regulation of
Msx-1, Msx-2, and Id3 mRNA--
We next wanted to assess to what
extent our results obtained in vitro using cultured cells
would also relate to gene induction events in vivo. The
dominant mouse mutant Fused toes (Ft) is
characterized by fused toes of the forelimbs and thymus hyperplasia
(41, 42). Recent experiments have shown a region of ectopic
Bmp4 misexpression in the anterior distal part of the
forelimbs of Ft embryos at day
E12.2 Therefore, we analyzed
the expression of Id3 and Msx-2 by whole mount
in situ hybridizations of limbs from heterozygous
Ft/+ embryos at day E12. Fig.
6 clearly reveals at the site of
strongest ectopic Bmp4 misexpression an increase in the
expression of Id3 mRNA. Msx-2 overexpression
was displayed in an extended region that stretched over the entire area
of Bmp4 misexpression (not shown). These embryonic
expression data strongly support the notion of Msx and
Id genes being targets for BMP4 signaling in
vivo.
Embryonic stem (ES) cells represent totipotent cells that can
contribute to normal embryonic development when incorporated into
blastocysts. In vitro, ES cells can also be stimulated to differentiate along mesodermal and neuronal paths, which has yielded significant insight into processes of cellular differentiation during
development. In the present study we confirm the ability of ES cells to
respond efficiently and specifically to extracellular differentiation-inducing signals. Furthermore, we used the ES cell
system to identify by dd-RT-PCR transcription units whose expression
was hitherto unknown to be stimulated upon treatment with the mesoderm
inducing factors BMP2 and BMP4. A similar experimental strategy led
previously to the identification of Cryptic as a novel
mesoderm-specific gene (61).
Expression of Transcriptional Control Genes upon BMP4 Treatment
of ES Cells
Msx Genes--
We confirmed the Msx-1 and
Msx-2 genes as target genes of BMP2/4 signaling in ES cells
(Fig. 1A and Fig. 2). Since Msx gene induction by
BMPs did not require de novo protein synthesis, it is
suggested that Msx-1 and Msx-2 represent direct
target genes of BMP signaling. Msx-1 and Msx-2
are two members of a divergent homeobox-related gene family displaying
homology to the Drosophila msh gene (muscle segment
homeobox) (reviewed in Refs. 62 and 63). In the mouse embryo
Msx genes are involved in epithelial-mesenchymal interactions (49, 64-66) and are expressed in a wide range of embryonic regions, including the neural tube, the limb buds, and derivatives of the cranial neural crest (67-70). Their overlapping expression patterns and sequence similarities strongly suggest that the
Msx-1 and Msx-2 genes function in an equivalent
manner during development (71). Since expression of Msx
genes correlates with that observed of BMPs during development, BMPs
have been considered likely candidates for Msx-inducing factors
(62).
Our identification of murine Msx genes as direct targets of
BMP signaling are in line with data recently published by Suzuki et al. (72) demonstrating in Xenopus embryos that
BMP receptor activity elicits Msx-1 induction and that
Msx-1 can mimic BMP effects in epidermal induction and the
inhibition of neural differentiation.
BMP activity and Msx gene expression have been identified as
part of certain apoptotic mechanisms (47). However, as judged by TUNEL
assays, our treatment of in vitro cultured ES cells with BMPs did not lead to enhanced apoptosis (data not shown).
fos and jun Genes--
The transcription factor AP-1 consists of
Fos/Jun heterodimers or Jun/Jun homodimers and activates gene
transcription programs in response to various stimuli, including
cytokines, growth factors, and cellular stress, like UV irradiation or
heat shock (44, 45). The data presented here show that the
c-fos gene did not significantly respond to treatment with
BMP4 (Fig. 1), whereas the c-jun transcript exhibited a
delayed and long lasting increase (Fig. 1B). JunB
mRNA levels were elevated very rapidly and transiently (Fig.
1B). This agrees with Chalaux et al. (73)
demonstrating JunB to be a direct BMP2 target in C2C12 cells
and to mimic the actions of BMP2 in inhibiting the expression of
myoblast differentiation markers. Additionally, Jonk et al.
(74) reported the identification of a regulatory cis-element
in the JunB promoter targeted by BMP signaling (74). The
prolonged response of the c-jun gene correlates with similar
effects elicited by TGF- Identification of Novel BMP-induced Immediate Early Genes by
Differential Display (dd) RT-PCR
Of the six BMP4-regulated immediate early genes revealed in this
study, five could be identified by their homology to known sequences.
These include genes encoding one extracellular matrix signaling
molecule (Cyr61/clone A52), one translational regulator (EIF4AII/clone
A59), and three transcriptional regulators (DEK/clone A51, Sp-1
homologue/clone C74, and Id3/clone G9).
Cyr61--
Transcriptional activation of the Cyr61 gene
by FGF, PDGF, and the BMP-related molecule TGF- ElF4A--
The RNA helicases eIF4AI and eIF4AII are components of
the translation initiation complex eIF4A, which facilitates translation of certain mRNAs (91). Murine EIF4AII expression shows
tissue-specific distribution (54). We speculate that induction of
EIF4AII in response to BMP stimulation might facilitate
translation of critical mRNAs present in ES cells.
DEK--
DEK, a nuclear protein, has site-specific DNA binding
activity that is likely involved in transcriptional regulation and
signal transduction (92). DEK transcripts are seen at high
levels from embryonic day 10 onward, and the gene is expressed
ubiquitously in different adult tissues (52).
Clone74--
The BMP-inducible gene corresponding to clone C74 was
previously identified as Glbp-23b, encoding a novel murine
Sp1-like protein (55). Its human homologue TIEG was
identified as a TGF- Clone A79--
The gene represented by clone A79 awaits identification.
Induced Expression of Id1, Id2, and Id3 upon BMP Treatment of
ES Cells
In addition to the transcripts discussed above, our screen
identified the Id3 gene to be activated by BMP2/4. We
further show that its relatives, Id1 and Id2,
also respond directly to treatment with BMP4. These inductions are seen
in ES cells, as well as a diverse range of cell lines. Id family
members encode negative regulators of the basic helix-loop-helix (bHLH)
transcription factors that have been found to play a central role in
the control of mammalian cell growth, differentiation, and
tumorigenesis (reviewed in Ref. 56). Id proteins lack the basic region
but possess the HLH motif. They can form heterodimers with the
ubiquitously expressed E proteins (E2A, E2-2, and HEB) and render them
inactive for DNA binding, dimerization with the tissue-restricted bHLH
proteins, and, consequently, activation of their target genes.
Down-regulation of Id genes is necessary for terminal
differentiation in many developmental processes, including myogenesis
(93, 94), myelopoiesis (95), lymphopoiesis (96), bone morphogenesis
(59), glomerular mesangial cell development (97), and trophoblast
development (98).
Cell cycle progression is also regulated by Id factors, in part via the
induction of the p21-encoding gene, which in itself represents a
BMP-regulated transcription unit (99, 100). Id proteins are viewed
generally as negative regulators of differentiation and as positive
regulators of proliferation. However, in our ES cell system
BMP-mediated enhancement of Id expression does not coincide with a
stimulation in DNA synthesis, as judged by thymidine incorporation
assays (data not shown).
During organogenesis the expression patterns of Id1, Id2,
and Id3 genes are highly overlapping with each other and
with the expression domains of BMPs. These sites of expression are
often associated with active mesenchymal-epithelial interactions (57, 58). During gastrulation Id1 and Id3 are
expressed in the embryonic ectoderm, and Id2 is expressed in
extraembryonic tissues, whereas Id4 expression cannot be
detected at this stage of development (101). This may suggest an
involvement of Id1, Id2, and Id3, but not of Id4, in gastrulation and
is in line with the absence of Id4 expression in ES cells
observed here. The characterization of Id1, Id2, and
Id3 as BMP-induced immediate early genes in ES cells may
implicate negative regulation of bHLH transcription factor activity to
accompany BMP-mediated inductive events during early embryogenesis.
BMP-mediated increase in Id expression may function as a
molecular switch for lineage specification by functionally blocking the
realization of developmental programs regulated by certain bHLH
transcription factors (i.e. myogenesis). In this way, the
realization of alternative programs (i.e. ventralization of
mesoderm, bone formation, etc.) may be initiated.
Misexpression of Bmp4 in Embryos Induces Ectopic Induction of Msx-2
and Id3
Previous work in chicken using BMP-releasing beads implanted at
selected embryonic sites has identified induced expression of
Msx-1 and Msx-2 in the vicinity of the beads (46,
50). We made use of the dominant mouse mutant Fused toes
(Ft) which is characterized phenotypically by fused toes of
the forelimbs, coinciding with ectopical misexpression of
Bmp4 in the anterior distal part of the forelimbs at
E12.2 Although we found expression of Bmp4 (102)
and Id3 (57) in wild-type limbs as described previously, at
sites of ectopical BMP4 misexpression in Ft/+ limbs we observed the
induced expression of Id3 (Fig. 6) as well as
Msx-2 (not shown). This suggests that activation of these
genes within the embryo is a direct consequence of local BMP
expression. This in vivo correlation of BMP expression and
target gene induction adds support to the experimental strategy of
identifying in ES cells BMP-regulated genes whose induced expression may be relevant not only inside ES cells but also within the embryo.
In conclusion, we propose that the direct target genes of BMP4
signaling identified here are involved in BMP-stimulated early processes of mammalian development. It will be of special interest to
compare the expression patterns of Msx-1, Msx-2,
c-jun, Id1, Id2, and Id3 in normal mice
versus those deficient in BMP2, BMP4, and BMP2/4-type I
receptor. The promoters of the genes identified in our study should
serve as useful tools to characterize the molecular gene regulatory
circuits that are governed by BMP signaling.
Recombinant BMP2 and BMP4 polypeptides were
gifts of Genetics Institute (Cambridge, MA); recombinant activin A was
provided by Genentech. cDNA clones were provided by F. Sablitzky
(Id1, Id2, and Id4), R. Deed, J. Norton
(Id3), V. Sukhatme (Egr-1), and R. Hill
(Msx-1, Msx-2). We acknowledge M. Wiles for providing CCE
cells. We thank Marianne Petri for excellent technical assistance and
Franziska Wiebel for comments on the manuscript.
*
This work was supported by the Deutsche
Forschungsgemeinschaft Grant SFB 271 and SFB 446 and the Fonds der
Chemischen Industrie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Institut für Biochemie und Biotechnologie,
Abteilung für Zellbiologie und Molekularbiologie, TU
Braunschweig, D-38106 Braunschweig, Germany.
¶
Present address: Institut für Genetik, Universität
Köln, D-50674 Köln, Germany.
2
J. Heymer and U. Rüther, manuscript
in preparation.
The abbreviations used are:
BMP(s), bone
morphogenetic protein(s);
CDM, chemically defined medium;
FCS, fetal
calf serum;
ES cells, embryonic stem cells;
CHX, cycloheximide;
TGF, transforming growth factor;
RT-PCR, reverse transcriptase-polymerase
chain reaction;
dd, differential display;
FCS, fetal calf serum;
bFGF, basic fibroblast growth factor;
PBS, phosphate-buffered saline.
Id Genes Are Direct Targets of Bone Morphogenetic
Protein Induction in Embryonic Stem Cells*
§,¶,,
, and**
Institut für Molekularbiologie,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-superfamily and have been identified in a wide variety of organisms, ranging from
insects to mammals (4). BMPs have originally been isolated for their
ability to induce ectopic bone formation when injected under the skin
or into the muscle of rodents (5, 6), but meanwhile many of the BMPs
have been implicated in a variety of other developmental interactions,
including very early embryonic inductive events. For example in
Drosophila embryonic dorsal-ventral patterning is partly
accomplished through the action of DPP (decapentaplegic) (7,
8), which is the BMP member most closely related to mammalian BMP2 and
BMP4. At the functional level these factors can substitute for one
another in vertebrate and Drosophila embryos. Human BMP4 is
able to rescue the dorsal-ventral pattern defects of dpp
null mutants (9), whereas Drosophila DPP protein can induce
ectopic bone in mice (10). The striking evolutionary conservation of
BMP2, BMP4, and DPP suggested that these molecules play crucial roles
in early vertebrate development, as is confirmed meanwhile by many
studies. In Xenopus laevis BMP4 ventralizes early mesoderm
(11-13) and promotes the differentiation of epidermis from ectoderm
(14). Blocking BMP2/4 receptor activity in the ventral part of the
embryo eliminates blood formation and dorsalizes the mesoderm (15, 16),
whereas in contrast overexpression of BMP4 RNA leads to an increased
expression of ventral genes and inhibits the formation of anterior
structures (11, 12, 17). Use of a dominant-negative BMP2/4 receptor to
block BMP signals in the ectoderm (18), or disaggregation of ectodermal cells (19), causes differentiation into neural tissues. Mutagenesis by
homologous recombination of the genes encoding BMP4 (20), BMP2 (21),
and the BMP2/4-RI receptor (22) have demonstrated the requirements for
BMP2 and BMP4 during early mouse development. Embryos homozygous for
the Bmp4 null mutation do not proceed beyond the egg
cylinder stage and show little or no mesodermal differentiation (20),
whereas mice deficient for BMP2 have defects in amnion/chorion and
cardiac development (21). Mice lacking the BMP2/4 type I receptor die
prior to gastrulation and fail to form mesoderm altogether (22). BMPs,
like other members of the TGF- superfamily, signal through
heteromeric complexes of type I and type II transmembrane Ser/Thr
kinase receptors (23, 24). Binding of BMP dimers to either type of
receptor triggers well defined signaling steps (25, 26) that finally
lead to the activation of crucial target genes. However, relatively
little is known about direct target genes of BMP signaling during early
mammalian cell fate determination. Therefore, the identification of
BMP-induced immediate early genes appeared of great interest. Toward
this aim we analyzed BMP-mediated gene activation in murine ES cells.
The in vitro differentiation process of ES cells can serve
as a model for early mammalian development, permitting the expression
of a variety of embryonic markers to be monitored (27, 28). When ES
cells are cultivated in a chemically defined medium (CDM), they exhibit
specific responses to defined growth factors. These cellular responses
are influenced by the nature as well as the concentration of the
factors. For example, BMP4 treatment induces expression of ventral
mesoderm markers (29), and, at higher BMP concentrations, hematopoietic
cell development is induced (30). We used this system to investigate BMP2 and BMP4 effects on the expression of early embryonic marker genes
by using the differential display (dd) RT-PCR technique (31). We report
the identification of several transcription units directly regulated by
BMPs, including members of the Id gene family.
EXPERIMENTAL PROCEDURES
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-monothioglycerol, 0.2 mM
L-glutamine, and antibiotics (50 units/ml penicillin, 50 µg/ml streptomycin). The medium was changed every day, and passaging was required every 2nd to 3rd day. The ES cell line E14.1
(129/Ola-derived) had been adapted to grow in the absence of feeder
cells (27, 34) and was maintained under the same conditions as the CCE cells.
32P]dATP. Washed membranes were
exposed to Kodak XAR films at 
80 °C using an intensifying screen.
To estimate RNA loading variations further, all filters were
subsequently rehybridized with a glyceraldehyde phosphate dehydrogenase
(Gapdh) cDNA probe (37). Where appropriate, quantification was performed by densitometry using the MacBas software
package (Fuji).
-33P]dATP, and aliquots of the
samples were resolved on 6% urea/acrylamide sequencing gels. Amplified
fragments were sized relative to the marker bands of PCR molecular
weight markers (Amersham Pharmacia Biotech) end-labeled with
polynucleotide kinase using [
-32P]ATP. The dried gels
were exposed to Kodak XAR films, and the autoradiographs were analyzed
for the presence of differentially displayed bands. Fragments of
interest were excised from the dried gel, eluted by soaking the gel
slice in 100 µl of dH2O for 10 min, followed by boiling
for 15 min. The DNA was precipitated using glycogen, sodium acetate,
and ethanol, and the precipitate was dissolved in a small volume of
dH2O. A portion of this was used for the reamplification
employing the same 5'- and 3'-primers used in the first PCR. The
reamplified fragments were analyzed in 1.5% agarose gels, excised,
purified by the Jetsorb extraction kit (Genomed GmbH) to remove dNTPs
and protein, and aliquots were used as probes for Northern blot analysis.
20 °C. After reprocessing through 50%
methanol, the embryos were bleached in 3%
H2O2/PBS, washed 3 times for 5 min in PBS, and
stored in hybridization mix until further usage. For further use, the
embryos were washed once for 5 min in PBS and were hybridized at
70 °C overnight with DIG-labeled riboprobes for Bmp4, Msx-2, and Id3
in 50% formamide, 5× SSC, 1% SDS, 1% Tween 20, 50 µg/ml heparin,
and 50 µg/ml Escherichia coli tRNA. Three
posthybridization washes were carried out in 50% formamide, 2× SSC,
and 1% Tween 20 at the hybridization temperature. Embryos were then
prepared for antibody incubation by processing once through PBS with
1% Tween 20 (PBT) followed by a 1-h incubation in 10% sheep serum in
PBT. Antibody incubation with an anti-DIG antibody (Roche Molecular
Biochemicals) was performed in 1% sheep serum in PBT (1:2000) at
4 °C overnight. After washing in PBT for at least 6 h the
embryos were incubated twice (20 min each) in detection buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2, 1% Tween 20) and stained in
detection buffer containing 20 µl nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate per ml stock solution
(Roche Molecular Biochemicals).
RESULTS
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DISCUSSION
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View larger version (39K):
[in a new window]
Fig. 1.
Signal-induced gene activation in ES cells
cultured in chemically defined growth medium. A,
expression of Msx genes (Msx-1, Msx-2)
and selected immediate early genes (c-fos, Egr-1)
in stimulated CCE cells. Cells were either grown normally (control
(C); lane 11) or treated with either BMP4 (5 ng/ml) (lanes 1-5) or bFGF (20 ng/ml) (lanes
6-10), harvested at the indicated time points after induction,
and 10 µg of extracted total RNA was applied to Northern analyses.
Gapdh rehybridizations are shown as RNA loading controls.
B, expression in CCE cells of Jun genes. For
control, mock treatments were for up to 24 h (lanes
1-6) or for 2 h (lanes 22 and 23), and
BMP4 treatments (10 µg/ml) were given for the indicated times
(lanes 7-21) or for 2 h (lanes 24 and
25). Additional pretreatment with cycloheximide
(CHX) (30 min) was given to some cells (lanes 23 and 25). Northern blotting was performed using probes for
c-jun (lanes 1-16 and 22-25) and
JunB (lanes 17-21 and 22-25).
View larger version (42K):
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Fig. 2.
Expression of the homeobox genes
Msx-1 and Msx-2 in BMP4-stimulated
CCE cells. A, BMP4-induced Msx expression
kinetics. Mock-treated CCE control cells (lanes 1-6) or
cells treated with BMP4 (10 ng/ml) (lanes 7-16) were
incubated for the indicated times, and total RNA was subjected to
Northern blot analysis. B, cycloheximide pretreatment of CCE
cells does not prevent BMP induction of Msx genes. Cells
were mock-treated (lanes 1 and 2) or were treated
for 2 h with BMP4 (10 ng/ml) (lanes 3 and
4). These cells were either preincubated with cycloheximide
(100 µg/ml) (lanes 2 and 4) or not preincubated
(lanes 1 and 3). RNA isolation, Northern
blotting, and hybridization with Msx probes and a
Gapdh control probe were performed as for Fig. 1.
View larger version (29K):
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Fig. 3.
Expression of genes, identified by
differential display, in BMP-stimulated CCE embryonic stem cells.
A, BMP4 induction of newly identified transcripts. CCE cells
were either mock-treated (control) or were incubated with BMP4 as
indicated. 15 min before these treatments half of the cultures received
actinomycin D (Act.D) (5 µg/ml). Northern blots were
performed as for previous figures, using as probes the cDNAs
indicated at the left of the figure. The obtained
BMP4-stimulated mRNA induction levels were quantitated, normalized
to Gapdh mRNA levels, and calculated relative to control
cell levels, and the obtained induction factors are indicated
below each panel. B, for comparison,
Id1 and Id2 genes are quantitated with regard to
their BMP4 induction in CCE cells.
View larger version (51K):
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Fig. 4.
Kinetic expression profiles of
Id1, Id2, and Id3
genes, and the effects of cycloheximide thereupon, in
BMP4-stimulated CCE cells. A, ES cells were
mock-treated (lanes 1-6) or were incubated with BMP4
(lanes 7-16) for the indicated times. Id gene
expression levels were analyzed by Northern blotting. The corresponding
control hybridizations with Gapdh are shown for quantitation
of RNA loading. B, BMP4-induced Id gene
activation in ES cells is resistant to treatment with cycloheximide.
Control (Contr.) cells (lane 1), and cells
treated with CHX (lane 2), BMP4 (lane 3), and
cycloheximide plus BMP4 (lane 4) are investigated by
Northern blotting. C, kinetic profile of BMP4-induced Id
protein expression (Western blotting).
View larger version (75K):
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Fig. 5.
Expression in different cell lines of the
Id1, Id2, and Id3
genes upon stimulation with BMP4. The indicated cell lines
were mock-treated or stimulated with BMP4 for 1-24 h, as indicated
(Time, h). Northern analysis and control hybridizations were
performed as outlined in previous figures.
View larger version (39K):
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Fig. 6.
Coexpression of BMP4 and
Id3 in mouse embryos. Whole mount in
situ analysis of Bmp4 and Id3 RNA expression
(Bmp4, upper row; Id3,
lower row) in forelimbs of wild type (wt, left
panel) and Ft/+ mutant (right panel) embryos
(E12.0). Parallel in situ hybridizations with other limb
expression markers (e.g. Shh and Pax6)
did not show any signal in the anterior distal regions of these mutant
embryo limbs (data not shown), thus demonstrating specificity of our
analysis. For orientation, the location of strongest Bmp4
misexpression is indicated by an arrow, with neighboring
regions displaying a gradient of Bmp4 misexpression.
DISCUSSION
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ABSTRACT
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REFERENCES
on human lung adenocarcinoma and
erythroleukemia cells, as well as mouse embryo fibroblasts (75-77).
The identification of c-jun and JunB as BMP
response genes in ES cells implicates AP-1 as a regulator of
BMP-dependent gene expression during early embryogenesis.
In this respect it is interesting to note that in mice null mutations
of the individual AP-1 components c-jun and JunB
lead to early embryonic death (78-80).
has been reported
previously (81, 82). The encoded Cyr61 protein is a secreted
cysteine-rich heparin-binding protein that associates with the cell
surface and the extracellular matrix (83). Cell-cell and cell-matrix interactions are known to exert important effects on embryonic development (84, 85). During embryogenesis cyr61 is
expressed most notably in mesenchymal cells that are differentiating
into chondrocytes and in vessels of the developing vascular system (86,
87). These sites overlap with the expression of BMP-related molecules
(88-90).
- and BMP2-regulated gene in osteoblasts (2).
The TIEG protein contains three zinc finger motifs homologous to the
transcription factors Sp1, Sp3, WT1, and GT box-binding protein. We
speculate that the C74 gene product may play a role as an effector of
BMP-mediated differentiation.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Entwicklungsbiologie und Molekularbiologie
der Tiere, Heinrich-Heine-Universität Düsseldorf, D-40225
Düsseldorf, Germany.
To whom correspondence should be addressed. Tel.: 49-7071-297 8898; Fax: 49-7071-29 53 59; E-mail:
alfred.nordheim@unituebingen.de.
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M. E. Bahamonde and K. M Lyons BMP3: To Be or Not To Be a BMP J. Bone Joint Surg. Am., March 1, 2001; 83(1_suppl_1): S56 - S62. [Abstract] [Full Text] [PDF]
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M. Schuldiner, O. Yanuka, J. Itskovitz-Eldor, D. A. Melton, and N. Benvenisty From the Cover: Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells PNAS, October 10, 2000; 97(21): 11307 - 11312. [Abstract] [Full Text] [PDF]
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