1 Department of Obstetrics and Gynecology, Faculty of Medicine, University of
Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
2 Department of Life Sciences, University of Tokyo, 3-8-1, Komaba, Meguro-ku,
Tokyo 153-8902, Japan
3 SORST Project, University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo 153-8902,
Japan
* Author for correspondence (e-mail: asashi{at}bio.c.u-tokyo.ac.jp)
Accepted 2 February 2003
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SUMMARY |
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Key words: Xenopus, FRL-1, EGF-CFC family, cripto, cryptic, one-eyed pinhead, Neural induction, Default model, Bone morphogenetic protein, Fibroblast growth factor, Ras/Raf/MAPK signaling
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INTRODUCTION |
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The Xenopus EGF-CFC protein FRL-1 was isolated as a ligand of the
fibroblast growth factor (FGF) receptor and is considered a member of the
EGF-CFC protein family (Kinoshita et al.,
1995; Shen et al.,
1997
). FRL-1 is expressed ubiquitously during
gastrulation and can induce neural and mesodermal markers in presumptive
ectoderm (Kinoshita et al.,
1995
). Studies of FGF signaling suggest the existence of two
signal transduction pathways mediated by the FGF receptor. First, activation
of the FGF receptor can activate the phospholipase C
(PLC
)
pathway to produce inositol triphosphate (InsP3) and
facilitate Ca2+ release (reviewed by Powers, 2000). Second, the FGF
receptor also signals to Ras, which subsequently activates the
mitogen-activated protein kinase (MAPK) signaling pathway (reviewed by Powers,
2000). It remains unclear which of these pathways interacts with FRL-1.
In vertebrate early development, bone morphogenetic protein (BMP) signaling
stimulates epidermal induction of undifferentiated cells in the presumptive
ectoderm region and inhibits neural induction
(Hemmati-Brivanlou and Melton,
1997). Neural cells are induced in the presumptive ectoderm when
BMP signaling is inhibited by factors such as Noggin, Chordin and Follistatin,
which are secreted from the organizer region that differentiates into axial
mesoderm. Initially, neural-inducing signals from the organizer are thought to
act in an instructive manner. They can, however, also bind directly to BMP
proteins. This leads to the `default model' of neural induction, which
proposes that in the absence of cell-cell signaling, ectodermal cells will
adopt a neural fate (Munoz-Sanjuan and
Hemmati-Brivanlou, 2002
).
In this study, we have examined FRL-1 function in early Xenopus development and find that it acts as a neural inducer. FRL-1 inhibits BMP signaling via the activation of MAPK signaling, implicating it early in neural induction. These data indicate that FRL-1-induced neural differentiation may occur via a nodal-independent mechanism.
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MATERIALS AND METHODS |
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Constructs
PBluescript FRL-1 (pBS-FRL-1) was obtained by screening
our cDNA library containing maternal genes. The construction of
pCS2-FRL-15'UTR was performed by amplifying the
FRL-1 ORF using PCR. For the construction of pCS2-FRL-1,
which contains the 5'UTR region, EcoRI- and
XhoI-digested pBS-FRL-1 was ligated into pCS2
vector. To test FRL-1 function (Figs
1,
10 and
11), we used
pCS2-FRL-1
5'UTR as FRL-1 because of its strong
activity. For construction of pCS2-FRL-1
5'UTR-6myc and
pCS2-FRL-1-6myc, FRL-1 cDNA fragments were amplified by PCR
from pCS2-FRL-1
5'UTR and pCS2-FRL-1,
respectively and the amplified products were ligated into pCS2+6myc vector.
pCS2-FRL-1
CFC-6myc and pCS2-FRL-1
EGF-6myc
constructs coded FRL-1-6myc protein without CFC domain (116Cys-stop codon),
and EGF domain (77Lys-112Arg), respectively. For construction of pCS2-oep,
oep ORF was amplified from pCDNA-oep-flag
(Zhang et al., 1998
) and
ligated into pCS2+ vector. For construction of pCS2-chickCFC,
pKS-chickCFC (Andree et al.,
2000
) was digested with PstI. The released fragments were
blunt-ended and ligated into pCS2 vector. For the preparation of mRNA,
pCS2-chordin, pCS2-lacZ
(Takahashi et al., 2000
),
pCS2-FRL-1, pCS2-FRL-1
5'UTR,
pCS2-FRL-1-6myc, pCS2-FRL-1
5'UTR-6myc,
pCS2-FRL-1
CFC-6myc, pCS2-FRL-1
EGF-6myc,
pCS2-cripto (Yeo and Whitman,
2001
), pSP64T-BMP-4, pSP64T-XFD
(Amaya et al., 1991
) and
pSP64T-SESE-MAPKK (Gotoh et al.,
1995
) were linearized and transcribed using the mMASSAGE mMACHINE
SP6 kit (Ambion).
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RT-PCR and histology
Total RNA isolation, RT-PCR and histological analyses were performed as
described previously (Tanegashima et al.,
2000). The primer pairs used were as follows; soxD
(forward, 5'-TCAGCAACAGGTCCAGTACC-3'; reverse,
5'-TCTAACAAGATCCGACGCC-3'), FRL-1 (forward,
5'-ATGCAGTTTTTAAGATTT-3'; reverse,
5'-TTAAAGTCCAATATT-3'), endodermin (edd) (forward,
5'-TATTCTGACTCCTGAAGGTG-3'; reverse,
5'-GAGAACTGCCCATGTGCCTC-3'), collagen type II
(col II) (forward, 5'-ATTCAGTTGACCTTCCTGCG-3'; reverse,
5'-TCCATAGGTGCAATGTCTACG-3'), Xngnr-1 (forward,
5'-CGCCGCAACCCGACTCACCT-3'; reverse,
5'-CCTGCATCGCGGGCTGTTCTC-3'), Xvent-1 (forward,
5'-TTCCCTTCAGCATGGTTCAAC-3'; reverse,
5'-GCATCTCCTTGGCATATTTGG-3'). The primer pairs for chordin,
Xmsx-1, ß-crystallin, ms-actin, EF1-
, otx2,
zic3, N-tubulin, Xbra and N-CAM were as described in Xenopus
Molecular Marker Resource
(http://www.cbrmed.ucalgary.ca/pvize/html/WWW/Welcome.html).
EF1-
was used as a loading control. Reverse transcriptase
negative (RT) reactions were performed to indicate the absence of
contaminating genomic DNA.
Whole-mount in situ hybridization
Analysis of whole-mount in situ hybridization was carried out as described
previously (Harland, 1991)
using digoxigenin-labeled antisense probes. For cell-lineage tracing, 20 ng of
FRL-1MO or 20 ng of FRL-1-4misMO were co-injected with 250 pg of lacZ
into one animal blastomere of albino embryos. Injected regions were stained
with red gal (Research Organics, Inc, USA). Probes were synthesized using
pBS-chordin (Sasai et al.,
1994
), pBS-zic3, pBS-soxD, pGEM-otx2,
pBS-Xngnr-1 (Ma et al.,
1996
) and pBS-FRL-1 as templates. PBS-soxD and
pBS-zic3 were obtained by our own screening.
Western blotting
FRL-1 proteins with 6myc-epitope tags were detected using the 9E10
monoclonal antibody (Santa Cruz). -actin served as the loading control
and was detected by the AC-40 monoclonal antibody (Sigma). Activated MAPK was
detected by monoclonal anti-MAPK, activated (diphosphorylated ERK-1 and 2)
clone MAPK-YT (Sigma) and total MAPK protein were detected by ERK2, rabbit
polyclonal IgG (Santa Cruz). Anti-mouse IgG, HRP-linked antibody (Cell
Signaling Technology, Inc.) and peroxidase-conjugated goat anti-rabbit IgG
(Sigma) were used as the secondary antibodies.
Whole-mount in situ immunohistology
Neu-1 monoclonal antibody was described previously
(Itoh and Kubota, 1989). After
detection, wild-type embryos with Neu-1 staining were bleached. For the
detection of activated MAPK, whole-mount in situ immunohistology was done
using monoclonal anti-MAPK, activated (diphosphorylated ERK-1 and 2) clone
MAPK-YT (SIGMA). The signal was detected using BM purple (Roche).
Proteins and chemical compounds for treatment
Purified bFGF (Amersham Biosciences), PD98059 (Biomolecules for Research
Success) and LY294002 (Biomolecules for Research Success) were purchased
commercially. PD98059 and LY294002 were dissolved in DMSO and stored at 10 mM
concentration.
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RESULTS |
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To test this hypothesis, we used the animal cap assay to examine whether
FRL-1 inhibited early BMP response genes induced by BMP-4
(Koster et al., 1991).
BMP-4 induced the expression of Xmsx-1, Xvent-1 and
Xbra at the early gastrula stage
(Fig. 1B, lane 2), whereas
FRL-1 completely inhibited the expression of BMP response genes
induced by BMP-4 (Fig.
1B, lane 3).
Under our experimental conditions, FRL-1 induced neural induction
without mesoderm induction at an early stage. We also tested whether
FRL-1 induced the expression of late neural markers and mesodermal
markers. FRL-1 induced the expression of N-CAM and the
anterior neural marker otx2
(Pannese et al., 1995) in a
concentration-dependent manner, but not ms-actin and collagen
type II (Su et al.,
1991
), which were expressed in muscle and notochord, respectively,
even when FRL-1 was used at high doses
(Fig. 1C). These results
indicate that FRL-1 can directly induce anterior neural tissue
without the induction of mesoderm.
EGF-CFC proteins show conserved function in neural induction
EGF-CFC proteins have two conserved motifs, the EGF-like and the CFC motif
(Shen and Schier, 2000). To
examine the function of these motifs in neural induction, two cDNA constructs
encoding an EGF-like motif-deleted form with the 6myc-epitope tag
(FRL-1
EGF domain) and a CFC motif-deleted form with the
6myc-epitope tag (FRL-1
CFC domain) were generated and used for
the animal cap assay. Western blotting confirmed expression of these protein
products in the injected animal caps. Overexpression of mRNA encoding FRL-1
with 6myc-epitope tag in ectoderm induced the neural marker N-CAM.
However, the FRL-1
EGF domain or FRL-1
CFC
domain showed no such inductive ability
(Fig. 2A). This suggests that
both the EGF-like and CFC domains of FRL-1 are required for neural
induction.
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FRL-1MO specifically inhibits translation of FRL-1
We clearly show that FRL-1 acts as a neural inducer by inhibiting
BMP signaling in the animal cap. To study the role of FRL-1 in neural
induction in Xenopus embryos, we generated an antisense morpholino
oligonucleotide against FRL-1 (FRL-1MO) and then tested whether it
specifically inhibited the translation of FRL-1 using the
Xenopus oocyte expression system
(Fig. 3). First, FRL-1-6myc
proteins, consisting of FRL-1 tagged with the 6myc-epitope at the C-terminal
region were detected at approximately 35 kDa and 25 kDa
(Fig. 3, lanes 2, 5). A 25 kDa
protein is consistent with the predicted molecular size of the FRL-1-6myc
protein. The 35 kDa band may represent a glycosylated form of FRL-1-6myc since
the mouse EGF-CFC protein, Cripto is known to be fucosylated
(Schiffer et al., 2001).
FRL-1MO was shown to inhibit the translation of FRL-1
(Fig. 3, lane 3), but not
FRL-1-4misMO, which contains four nucleotide substitutions to exclude the
toxicity of MO or FRL-1
5'UTR that lacks the target
sequence of FRL-1MO (Fig. 3,
lanes 4, 6). These results imply that the FRL-1MO specifically inhibited FRL-1
translation.
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To further examine whether MAPK activation is required for FRL-1 regulation
of neural induction, we used a MEK inhibitor, PD98059, to inhibit MAPK
activation by MEK. In animal caps, the expression of N-CAM induced by
the overexpression of FRL-1 was inhibited by treatment with PD98059,
suggesting that FRL-1 is required for MAPK activation to induce the
expression of neural markers (Fig.
11A). In contrast, a specific inhibitor of PI3K, LY294002 did not
affect the N-CAM induction by FRL-1
(Fig. 11A), even though the
FGF receptor is known to activate PI3K signaling
(Powers et al., 2000). We also
showed that FRL-1 acts as a neural inducer by inhibiting BMP
signaling. We therefore examined whether inhibition of BMP signaling by
FRL-1 was affected by the MEK inhibitor, PD98059
(Fig. 11B). RT-PCR analysis
showed that the expression of BMP responsive genes, Xmsx-1 and
Xvent-1, which was suppressed by FRL-1 in animal caps, were
rescued by treatment with PD98059 (Fig.
11B). These data suggest that inhibition of BMP signaling by FRL-1
is required for MAPK activation.
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DISCUSSION |
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FRL-1 suppresses BMP signaling to regulate an early step of
neural induction
The expression of early neural genes such as soxD, sox2, and
zic-related genes is activated in the presumptive neuroectoderm of
gastrulae, and signals the start of neuralization
(Sasai, 2001). In animal cap
assays, we now show that overexpression of FRL-1 induces
soxD and zic3, and FRL-1MO inhibits the induction of these
genes in neuroectoderm, suggesting that FRL-1 regulates an early step
of neural induction. Our studies also suggest that FRL-1MO-injected ectoderm
loses competence to respond to the neural inducer, chordin, which
acts as an antagonist of BMP. In Xenopus, the `default model'
proposes that neural induction occurs only by BMP inhibition in the
presumptive ectoderm (Munoz-Sanjuan and
Hemmati-Brivanlou, 2002
). However, in chick, misexpression of
chordin does not induce the expression of neural markers in
non-neural ectoderm (Streit et al.,
1998
), but does induce Hensen's node, which is equivalent to the
Xenopus organizer (Streit et al.,
1998
; Streit et al.,
2000
). These observations indicate that the inhibition of BMP
signaling is not sufficient for neural induction and extracellular factors are
required in Hensen's node in chick. FGFs are good candidates since FGFs induce
neural tissue (Storey et al.,
1998
; Wilson et al.,
2000
) and the FGF receptor inhibitor SU5402 inhibits early neural
induction in chick (Streit et al.,
2000
). In Xenopus, FGFs also induce neural tissue
(Kengaku and Okamoto, 1993
;
Lamb and Harland, 1995
;
Launay et al., 1996
) and
dominant-negative mutants of FGF receptor type 4a efficiently inhibit anterior
neural tissue (Hongo et al.,
1999
). Although FGF signaling is therefore required in both
Xenopus and chick, FGFs induce only posterior neural tissue,
suggesting that other ligands are responsible for inducing anterior neural
tissue. We propose that EGF-CFC proteins are strong candidate neural inducers
that can activate FGF signaling. We showed that FRL-1 induces the
expression of anterior neural markers and that FRL-1MO inhibits the induction
of anterior neural tissue. In addition, FRL-1 activates the FGF receptor/MAPK
signaling pathway. We note that the expression of chick CFC is
condensed in Hensen's node region (Colas
and Schoenwolf, 2000
), in contrast to the ubiquitous expression of
Xenopus FRL-1 (Kinoshita et al.,
1995
). The expression patterns of chick CFC may
correspond to a neural inducer in Hensen's node and may reflect the different
competence in responding to neural inducers between Xenopus and
chick.
FRL-1 inhibits BMP signaling via the activation of FGFR/MAPK
signaling
Although it has been shown that FRL-1 activates FGFR-dependent
Ca2+ release in oocytes and that XFD inhibits FRL-1 function in
animal caps, we show here that FRL-1 activates MAPK in blastula embryos and
its activation is required for BMP inhibition and neural induction by FRL-1.
Furthermore, we showed both the EGF and CFC domains are necessary for neural
induction of FRL-1 (Fig. 2A). However, the EGF-like domain alone of the EGF-CFC protein Cripto is sufficient
to activate the MAPK pathway in mouse mammary epithelial cells
(Kannan et al., 1997). This
difference may be explained by these proteins binding different receptors:
FRL-1 function may depend on FGF receptor signaling
(Kinoshita et al., 1995
)
(Fig. 10A), whereas in mouse
mammary epithelial cells, human Cripto indirectly activates ErbB4
(Kannan et al., 1997
;
Bianco et al., 1999
). These
results suggest that both the EGF and CFC domains may be required for FGF
receptor-mediated functions of EGF-CFC proteins.
While it is known that EGF-CFC genes are involved in nodal signaling, in
this study, overexpression of FRL-1 inhibited BMP signaling in animal
caps, where nodal-related genes are not expressed, suggesting that
BMP signaling was blocked in the absence of nodal signaling. We also found
that oep, mouse cripto and chick CFC induced the
expression of neural markers in Xenopus animal caps. Our observations
suggest that BMP signaling may not only be inhibited by FRL-1 but
also by EGF-CFC genes in a nodal-independent fashion. Although EGF-CFC
proteins are required for nodal signaling, the mouse EGF-CFC protein Cripto
activates MAPK signaling, independent of nodal and its receptor, ALK-4
(Bianco et al., 2002). These
results suggest that EGF-CFC protein is able to act via MAPK activation, which
is independent of nodal. Previous studies showed that activated Ras
could not rescue oep mutant in zebrafish
(Gritsman et al., 1999
),
suggesting that Oep function is not required for MAPK activation. oep
mutants have defects in mesendodermal tissue and are therefore thought to lack
a cofactor for nodal. Our results showed that FRL-1 acts via a MAPK pathway in
neural induction (Fig. 11A).
These results suggest that a functional EGF-CFC protein is required for MAPK
activation in neural induction but not in nodal signaling.
It is known that some extracellular ligands that activate MAPK signaling
have opposite effects to BMP. For example, FGF opposes the anti-proliferative
effect of BMP-2 during limb bud outgrowth
(Niswander and Martin, 1993)
and epidermal growth factor (EGF) antagonizes the induction of osteogenic
differentiation markers by BMP-2 (Bernier
and Goltzman, 1992
). Previous reports have suggested that
MAPK-mediated phosphorylation negatively regulates the function of Smad1, an
intracellular mediator of BMP signaling
(Kretzschmar et al., 1997
).
MAPK phosphorylates the linker domain of Smad1, thereby inhibiting its nuclear
translocation and subsequent BMP signaling. This result seems to be applicable
for FRL-1 function in inhibiting BMP signaling and sustaining neural
competency. Furthermore, Sater and colleagues showed that MAPK activation is
required for the induction of Xenopus neuroectoderm
(Uzgare et al., 1998
;
Goswami et al., 2001
). Their
results suggest that upregulation of MAPK activity is detected in the
neuroectoderm of dominamt negative BMPR- or noggin-injected
ectoderm and that the overexpression of MAPK phosphatase, which inactivates
activated MAPK, inhibits neural induction in Xenopus
(Goswami et al., 2001
). Our
results could account for upregulation of MAPK activity by the observed
enrichment of FRL-1 transcripts in neuroectoderm and the induction by
chordin. Conversely, it was proposed that BMP signaling might inhibit
MAPK activity via the TAK1 pathway
(Goswami et al., 2001
),
indicating mutual antagonisms. Taken together, our results suggest that
antagonistic effects between the FRL-1/FGFR/MAPK pathway and BMP signaling are
involved in the establishment of neural versus epidermal cell fate.
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ACKNOWLEDGMENTS |
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