Department of Biological Sciences, Graduate School of Science, University of Tokyo, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
* Author for correspondence (e-mail: m_taira{at}biol.s.u-tokyo.ac.jp)
Accepted 16 January 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Xenopus laevis, Neuralization, BMP signaling, Smad, Inner nuclear membrane protein, MAN1
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is widely accepted that inhibition of BMP signaling triggers neural
induction in Xenopus embryogenesis. In recent years, a basic model
for the signal transduction pathway from BMP receptors to the nucleus has been
established (Heldin et al.,
1997; Whitman,
1998
; Massague and Chen,
2000
; Wrana,
2000
). BMP type II receptors form hetero-oligomeric complexes with
type I receptors upon binding to BMPs. Subsequently, type II receptors
phosphorylate and activate type I receptors, which, in turn, phosphorylate
Smad1, Smad5 or Smad8. Phosphorylated Smads associate with Smad4 and
translocate to the nucleus, where the complex binds to target genes together
with an appropriate transcription factor(s). Xvent1, Xvent2, Msx1 and
Xhox3 have been identified as BMP target genes in early
Xenopus embryogenesis. In the case of Xvent2, OAZ has been
identified as a Smad1-interacting transcription factor mediating the
upregulation of Xvent2 by BMP in the ventral region of
Xenopus gastrulae (Hata et al.,
2000
).
Finding the molecules that modulate this signaling has helped our
understanding of the mechanisms of neural induction by BMP antagonism
(von Bubnoff and Cho, 2001).
These molecules include extracellular antagonists (the Noggin, Chordin,
Follistatin, and DAN family molecules), which bind BMPs to prevent activation
of BMP receptors (Sasai et al.,
1995
; Zimmerman et al.,
1996
; Fainsod et al.,
1997
; Hsu et al.,
1998
; Iemura et al.,
1998
; Piccolo et al.,
1999
), and a pseudoreceptor (BAMBI), which acts as a
dominant-negative BMP receptor
(Onichtchouk et al., 1999
).
Molecules that interfere with Smad functions are also involved in BMP
antagonism. Smad6 blocks the BMP pathway by competing with Smad4 for binding
to activated Smads (Hata et al.,
1998
), and Smad7 inhibits phosphorylation of Smad1 by interacting
with activated BMP type I receptors
(Souchelnytskyi et al., 1998
).
The Ski oncoprotein and Smad-interacting protein (SIP) bind to Smad1
(Verschueren et al., 1999
;
Wang et al., 2000
) and
suppress the expression of BMP target genes
(Wang et al., 2000
). Smurf1
and Smurf2, which are ubiquitin E3 ligases, mediate ubiquitination and
degradation of Smad1 (Zhu et al.,
1999
; Zhang et al.,
2001
). Inhibition of the BMP pathway achieved by these molecules
leads to neuralization of animal explants in Xenopus
(Casellas and Brivanlou, 1998
;
Nakayama et al., 1998
;
Eisaki et al., 2000
;
Harland, 2000
), and is also
thought to be crucial for controlling the expression levels of target genes
for BMP signaling.
A Xenopus expression cloning system has been one of the most
powerful methods used to identify molecules that are involved in a variety of
developmental processes (Grammer et al.,
2000). Although many novel genes have been successfully identified
with this system, most of this work has been carried out using cDNA libraries
prepared from whole embryos containing different cell populations, which
results in the dilution of gene contents of interest and hence increases in
the number of genes needed to be screened. We therefore made an anterior
neuroectoderm (ANE) cDNA library from Xenopus late gastrulae to
increase cloning efficiency, and screened it for genes that are involved in
neuralization and neural patterning. We focused on the ANE to clarify the
following features of ANE: (1) it might contain a source of secreted factors
responsible for `homeogenetic induction', neural induction by the neural plate
(Mangold and Spemann, 1927
;
Servetnick and Grainger,
1991
); (2) the ANE should have its own neuralizing activity as a
default state after suppressing BMP signaling; and (3) the ANE may have
self-regionalizing activity to give rise to brain structures from the
forebrain to midbrain, and thus generate a wide variety of cell populations
during neurulation.
To date, several inner nuclear membrane proteins are known, including lamin
B receptor (LBR), lamina-associated polypeptide 1 (LAP1), LAP2, emerin, MAN1
and nurim (Worman and Courvalin,
2000). Most of these have been shown to interact with the nuclear
lamina and chromatin. In addition, LAP2, emerin and MAN1 share a conserved
domain, called the LEM domain, near their N termini
(Lin et al., 2000
). The LEM
domains of LAP2 and emerin have been shown to bind barrier-to-autointegration
factors (BAFs) (Furukawa,
1999
; Lee et al.,
2001
; Shumaker et al.,
2001
), which bind to DNA to bridge between the LEM
domain-containing protein and DNA, and are thought to be involved in chromatin
decondensation and nuclear assembly
(Segura-Totten et al., 2002
).
However, the physiological functions of inner nuclear membrane proteins have
not been fully elucidated, especially in terms of signal transduction
pathways.
We report here a novel neuralizing factor, XMAN1, the ortholog of human MAN1, identified by an expression screening with the ANE cDNA library. We also present evidence that XMAN1 acts as a Smad-interacting protein and could be involved in neural development by antagonizing BMP signaling. This provides new insights into the molecular mechanisms of neural induction and the regulation of BMP signaling, as well as elucidating the role of an inner nuclear protein in gene regulation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction and functional screening of an ANE library
About 500 pieces of anterior neural plates were dissected from stage
12-12.5 embryos. The neuroectoderm layer was separated from the underlying
mesendoderm layer in 1x modified Barth's solution containing 1-2 mg/ml
collagenase (Shibata et al.,
2001). After poly (A)+ RNA selection by an oligo(dT)
cellulose column, a cDNA library was made with the SuperScript plasmid system
for cDNA synthesis and plasmid cloning (Invitrogen) and the pCS105 vector. Two
hundred pools, each containing about 200 independent cDNA clones, were
prepared, and capped RNA was synthesized from each pool as previously
described (Taira et al.,
1994
). Capped RNA (10 ng) was injected into the animal pole region
of one-cell stage embryos. Animal caps were dissected at stages 8-9 and
cultured until siblings reached stage 25.
cDNA cloning and plasmid constructs
Screening of a stage 30 head cDNA library for a full-length clone of
XMAN1 was carried out using a standard method. 5'-RACE was
performed using the 5'-full RACE core set (Takara) and
poly(A)+RNA from stage 12.5 embryos. Constructs for Myc-tagged
full-length (amino acids 1-781, MT-XMAN1), LEM (amino acids 44-781,
MT-
LEM),
NT (amino acids 450-781, MT-
NT),
CT
(amino acids 1-581, MT-
CT) and CT (amino acids 582-781, MT-CT) of XMAN1
were made using PCR and the pCS2+MT vector. MT-
RNP1, MT-
RNP2 and
MT-
RNP1+2 constructs were made with the GeneEditor in vitro
site-directed mutagenesis system (Promega) using MT-XMAN1 as a template.
HA-tagged mouse Smad3, Smad5, Smad6, Smad7 and Smad8 were made by transferring
the inserts from pcDEF3 constructs to the pCS2+HA vector.
Whole-mount in situ hybridization and histological studies
Whole-mount in situ hybridization was performed according to Harland
(Harland, 1991) using an
automated system (AIH-101, Aloka). An antisense XMAN1 RNA probe was
synthesized by transcribing a NotI-linearized clone pXMAN1-11 (the
longest clone found after library screening) with T7 RNA polymerase. Other RNA
probes were synthesized according to plasmid providers. Some stained embryos
were embedded in paraffin wax and sectioned at 10-15 µm.
RT-PCR
Animal caps and marginal explants were dissected when siblings reached the
stages indicated, and RT-PCR was carried out as previously described
(Osada and Wright, 1999),
except that DNA amplification was achieved non-radioactively. PCR products
were analyzed on 2% agarose gels in the presence of ethidium bromide; images
of fluorescent DNA fragments were digitally recorded by GelPrint 2000i
(Genomic Solutions), and black/white inversion was performed using PhotoShop
software (version 5.5, Adobe Systems). Primers for XMAN1 were: forward,
5'-CAAATTTGCAGTCATGCTCT-3'; reverse,
5'-AAAATAAGTGGGGCCCTATG-3'. In several experiments, real-time
RT-PCR was carried out using ABI PRISM 7000 (Applied Biosystems) with Taq-Man
probes (Table 1) that allowed
the detection of only primer-specific PCR products. Each real-time PCR was
performed in triplicate. EF-1
was used as an internal control and each
bar was normalized to the level of EF-1
expression.
|
Immunofluorescence microscopy
COS-7 cells were grown in Dulbecco's modified Eagle's (DME) medium
containing 10% fetal bovine serum. For transfection, cells were grown to
almost 80% confluence and transfected using FuGENE 6 Transfection Reagent
(Roche). After 24-hour incubation, transfected cells were washed twice with
phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde in PBS for
15 minutes at room temperature. Fixed cells were then permeabilized with 0.5%
Triton-X 100 in PBS for 10 minutes, blocked in PBS containing 10% lamb serum
for 1 hour at room temperature, and incubated with primary antibody in
Tris-buffered saline (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.01%
Tween 20 (TBS-T) for 1 hour at 37°C. Anti-FLAG M2 monoclonal antibody
(Sigma) and anti-Myc monoclonal antibody 9E10 (Biomol) were used as primary
antibodies at a dilution of 1:1000. After washing three times with TBS-T, the
cells were incubated with secondary antibody Alexa 488-conjugated goat
anti-mouse IgG (Molecular Probes) at a concentration of 5 µg/ml. Nuclei
were visualized by co-transfecting the pDsRed2-Nuc vector (Clontech). Protein
localization was examined by laser-scanning confocal microscopy using LSM5
Pascal (Zeiss).
GST pull-down assays
The C-terminal region of XMAN1 was subcloned into the pGEX-6P-1 vector to
produce the GST-XMAN1-CT fusion protein. [35S]methionine-labeled
proteins were synthesized with the TNT SP6-coupled reticulocyte lysate system
(Promega). Purification of GST and GST-XMAN1-CT proteins, and GST pull-down
assays were as previously described
(Hiratani et al., 2001).
Western blotting and immunoprecipitation experiments
Capped RNAs for Myc-tagged XMAN1 constructs were co-injected with or
without that for HA-tagged XSmad1 into the animal regions of two-cell stage
embryos. Injected embryos were cultured until the mid gastrula stage 11,
homogenized in 1x lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5
mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, 1 mg/ml aprotinin; 100 µl per
embryo), and microcentrifuged. The lysate (250 µg protein) was incubated
with 1 µg of anti-HA polyclonal antibodies (Santa Cruz) for 1 hour at
4°C, then incubated with protein G-agarose (Roche) for another hour.
Immunoprecipitates were washed five times in 1x lysis buffer, boiled in
SDS sample buffer, and analyzed by western blotting with anti-Myc antibody
9E10 (1:2000, BIOMOL) and horseradish peroxidase-conjugated anti-mouse IgG
antibody (1:2000, Amersham Pharmacia). Probing with a monoclonal
anti-ß-tubulin antibody (1:2500, Sigma) was for loading control.
Detection was carried out using the ECL+Plus kit (Amersham Pharmacia).
Antisense morpholino experiments
Antisense morpholino oligos complementary to XMAN1 mRNA (XMAN1-MO,
5'-GCCGCCATTTTGACCACTCGGT-3'), its four-base-mismatched control
oligos (XMAN1-4mmMO, 5'-GCgGCgATTTTGACCAgTCcGT-3'; where lower
cases indicate mismatch mutations) and nonspecific control oligos were
obtained from Gene Tools.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
XMAN1 dorsalizes the ventral mesoderm
Several neuralizing factors can induce a secondary axis when expressed in
the ventral marginal zone. Fig.
5A shows that this is also the case for XMAN1. XMAN1-CT induced a
secondary axis (parts b,d) that contained muscle, but not the notochord (part
f and data not shown). We next examined dorsal marker gene expression by
ventral marginal zone assays. In XMAN1-CT mRNA-injected marginal zones, all
the dorsal markers (gsc, chordin and -actin) were
elevated, whereas the ventral markers (Xhox3, Msx1 and
Xvent1) were suppressed, and the level of a pan-mesodermal marker
(Xbra) was unchanged (Fig.
5B). These results indicate that XMAN1-CT dorsalizes the ventral
mesoderm without changing the fate of the mesoderm.
|
|
To investigate whether XMAN1 blocks the signal transduction of BMP, we co-injected mRNA for XMAN1 with that for BMP4 or an activated form of type I BMP4 receptor, Alk3*. BMP4 or Alk3* alone induced the downstream targets of BMP signaling, Xhox3 and Msx1 (Fig. 6D). The induction of these markers was inhibited by XMAN1-CT in a dose-dependent manner, whereas the induction of Xbra by BMP4 and Alk3* was less sensitive to the inhibitory effects of XMAN1-CT. XMAN1-CT at higher doses (more than 500 pg per embryo) and wild-type XMAN1 at moderate doses (100-250 pg per embryo) almost completely inhibited BMP-induced Xbra expression (not shown). These results indicate that XMAN1 antagonizes BMP4 signaling downstream from the receptor.
The above results were supported by a reporter assay using a BMP-responsive
Xvent2-luc construct (Fig.
6E). Because BMP proteins are present in ectodermal explants
(Hemmati-Brivanlou and Thomsen,
1995), Xvent2-luc is activated at a certain level without
stimulation with BMP4 or Alk3*. Injection of BMP4 or Alk3* mRNA
enhanced this basal level by two- to threefold. By contrast, co-expression of
the full-length or the C-terminal constructs of XMAN1 (MT-XMAN1 or MT-CT), but
not a C-terminal truncated construct (MT-
CT), suppressed the reporter
activity even below the basal level. Repeatedly, MT-XMAN1 appears more
efficient in suppressing Xvnet2-Luc than MT-CT, suggesting that proper
intracellular localization is necessary for the full activity of XMAN1 (see
Fig. 4D).
XMAN1 binds to BMP-responsive Smads
Several Smad-interacting proteins have been shown to interfere with
TGFß signaling pathways (Heldin et
al., 1997; Massague and Chen,
2000
; Wrana,
2000
). Thus, we next examined whether XMAN1 physically associates
with BMP-responsive Smads by co-immunoprecipitation and GST pull-down assays.
MT-XMAN1, MT-
CT or MT-CT was co-expressed in embryos with HA-tagged
Xenopus Smad1 (XSmad1), a transducer of BMP signaling. XSmad1 was
then immunoprecipitated with an anti-HA antibody, and subsequently the
precipitates were analyzed by western blotting with an anti-Myc antibody. As
shown in Fig. 7A, MT-XMAN1 and
MT-CT, but not MT-
CT were co-immunoprecipitated with XSmad1, indicating
that XMAN1 interacts with XSmad1 through its C-terminal region in the embryo.
MT-CT was also co-immunoprecipitated with endogenous XSmad1
(Fig. 7B).
|
XMAN1 activity in neural development
To analyze the role of endogenous XMAN1, we suppressed XMAN1 activity by
injecting antisense morpholino oligos (XMAN1-MO) complementary to the sequence
around the initiation codon. As controls, we injected nonspecific control
morpholino (CTL-MO) and four-base-mismatched morpholino oligos (XMAN1-4mmMO).
We first examined the specificity of XMAN1-MO using an XMAN1 construct in
which the 5'UTR is retained to hybridize with XMAN1-MO and which encodes
Myc-tags at the C terminus (referred to as 5'UTR-XMAN1-MT). Co-injection
of XMAN1-MO inhibited the translation of 5'UTR-XMAN1-MT mRNA at the
early gastrula stage (stage 10.5), but co-injection of XMAN1-4mmMO did not
(Fig. 8A). This inhibitory
effect of XMAN1-MO on the translation of 5'UTR-XMAN1-MT mRNA was also
observed at the tailbud stage (stage 25). The specificity of XMAN-MO was
further examined by real-time PCR with animal caps. Injection of
5'UTR-XMAN1-MT mRNA induced nrp1 expression in animal caps as
does MT-XMAN1 mRNA that lacks the annealing sequence for XMAN1-MO
(Fig. 8B). Co-injection of
XMAN1-MO suppressed the expression of nrp1 induced by
5'UTR-XMAN1-MT mRNA but not by MT-XMAN1 mRNA, indicating that XMAN1-MO
specifically suppresses the neuralizing activity of 5'UTR-XMAN1-MT.
|
Because XMAN1 antagonizes BMP signaling as mentioned above, suppression of
XMAN1 activity by XMAN1-MO might lead to upregulation of BMP activity and
hence causes eye defects as shown above. To address this possibility, we
examined the expression of Dlx3, an epidermal marker
(Feledy et al., 1999), in
XMAN1 morphants. As shown in Fig.
8D, the Dlx3-negative region corresponding to the future neural
plate was shortened and narrowed particularly in the anterior region at the
early neurula stage 13 (n=18/20). The expression domain of
Msx1, another epidermal marker and a direct target of BMP signaling
(Suzuki et al., 1997
), was
also expanded in XMAN-morphants (n=8/8, not shown). In addition,
expression domains of Rx2A (retina)
(Mathers et al., 1997
),
Xemx1 (dorsal telencephalon)
(Pannese et al., 1998
),
nrp1 (Knecht et al.,
1995
) and Xsix3 (forebrain)
(Zhou et al., 2000
) were
severely reduced (Fig. 8Ci;
Rx2A, n=18/20; Xemx1, n=4/6; nrp1, n=8/9;
Xsix3, n=10/10, not shown). When XMAN1-MO was injected into all
animal blastomeres at the four-cell stage, development of anterior structures
was also severely reduced. Furthermore, when XMAN1-MO was injected dorsally at
the four-cell stage, similar phenotypes were observed but expression of dorsal
markers, goosecoid and chordin, was not affected (not
shown). These data imply that the XMAN1-MO phenotype is caused by a decrease
in XMAN1 activity in the anterior neuroectoderm where XMAN1 is expressed, and
not by a reduction in the dorsal mesoderm. Taken together, these results
suggest that the XMAN1 function is required for anterior neural
development.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We presume that the inhibitory action of XMAN1 to BMP signaling is due to
its binding specificity with the BMP-responsive Smads. XSmad1 interacts with
XMAN1 via its MH2 domain, to which several transcription factors,
co-activators and co-repressors bind
(Massague and Chen, 2000;
Wrana, 2000
). Therefore, XMAN1
may compete with those transcription factors or coactivators for binding to
the MH2 domain, or it may recruit a co-repressor on the MH2-domain as has been
suggested for the Ski oncoprotein (Wang et
al., 2000
). To address these questions it would be helpful to find
other binding proteins to the XMAN1/Smad1 (or Smad5, Smad8) complex.
The presence of the LEM domain in XMAN1 implies that XMAN1 on the inner nuclear membrane interacts with BAFs through the LEM domain and thus affects DNA indirectly. However, the role of the LEM domain in inhibiting BMP signaling is still unclear, because deletion of the LEM domain did not affect the activity of XMAN1 in neuralizing ectoderm (Fig. 4C).
Interactions between XMAN1 and XSmad1
Strong structural similarity of XMAN1 to human MAN1 enabled us to deduce
that XMAN1 localizes in the inner nuclear membrane. This is supported by
confocal immunofluorescent microscopy in which the subcellular localization of
XMAN1 is very similar to that of emerin, a known inner nuclear membrane
protein (Manilal et al., 1996;
Nagano et al., 1996
). Given
that XMAN1 localizes to the inner nuclear membrane, how does it regulate BMP
signaling? According to a current model, the C-terminal region of human MAN1
is likely to face the nucleoplasm (Lin et
al., 2000
). Thus, in the simplest way, activated Smad1 or
Smad1/co-factor/transcription complex in the nucleus may be trapped and
targeted to the inner nuclear membrane through binding to the C-terminal
region of XMAN1, leading to disruption of the transcription complex on BMP
target genes. Actually, several transcription factors have been shown to
become inactive when they are localized to the nuclear envelope
(Cohen et al., 2001
). However,
XMAN1-CT, which lacks the transmembrane domains necessary for its nuclear
membrane localization (Wu et al.,
2002
) and is ubiquitously distributed within the cell
(Fig. 4D), still showed
neuralizing activity (Fig. 4C).
This implies that the physical interaction between XSmad1 and XMAN1-CT
irrelevant to subcellular localization is enough to antagonize the BMP pathway
under our experimental conditions. Nevertheless, we have noticed that
full-length XMAN1 reproducibly shows stronger activity than XMAN1-CT in
suppressing the expression of BMP downstream targets in animal caps and
Xvent2-Luc activation (Fig.
6E), suggesting that some domains outside the C-terminal region or
nuclear localization of XMAN1 are necessary for the full activity.
In the case of the TGF-ß/activin-responsive Smads, Smad2 and Smad3, as
well as Smad4, it has recently been shown that nucleocytoplasmic shuttling of
those Smad proteins occurs constitutively
(Inman et al., 2002;
Xu et al., 2002
), and that
this process for Smad2 is conducted by the nucleoporins, CAN/Nup214 and Nup153
(Xu et al., 2002
). The balance
of those Smads between the cytoplasm and the nucleus is thought be maintained
by the cytoplasmic retention factors (SARA, microtubules and the actin binding
protein filamin) and Smad-interacting transcription factors in the nucleus,
such as FAST-1 (Tsukazaki et al.,
1998
; Dong et al.,
2000
; Sasaki et al.,
2001
; Xu et al.,
2002
). Phosphorylation of Smad2 shifts the balance to increase the
nuclear pool of Smad2. However, it has not been clarified whether
BMP-responsive Smads are regulated by similar mechanisms, while both the
nuclear localization signal (NLS) and the nuclear export signal (NES) have
been identified in Smad1 (Xiao et al.,
2001
). The association of XMAN1 and Smad1/Smad5/Smad8 may regulate
the amount of Smads accessible to downstream target genes at the nuclear
membrane level.
RRM in XMAN1
We found an RRM in the C-terminal region of XMAN1 that had not been
described before. Interestingly, this RRM is required for the neuralizing
activity of XMAN1 (Fig. 4C).
Because RRMs are frequently found in RNA-binding proteins and mediate their
association with RNAs (Birney et al.,
1993), it might be possible that RNAs mediate the neuralizing
activity of XMAN1. However, in our immunoprecipitation experiments, RNase
treatment of the cell lysate did not abolish the interaction of XMAN1-CT and
XSmad1 (data not shown). In addition, GST pull-down assays show that XMAN1 and
BMP-responsive Smads can interact directly in vitro
(Fig. 7C). These results
suggest that the association between the two proteins is mediated by
protein/protein interaction rather than via RNA, although we cannot exclude
the possibility that some RNA may interact with XMAN1 to modify BMP signaling
or to exert an unidentified role. RRM functions as a protein/protein
interaction domain as exemplified by PTB-associated splicing factor
(Dye and Patton, 2001
).
Involvement of the RRM in binding to Smads has not been reported before. We
could not find any sequence similarity between the RRM of XMAN1 and known
Smad-interacting motifs found in some Smad-binding proteins, such as OAZ,
Smurf1, FAST1 and Mixer, and phenylalanine-glycine (FG) repeats required for
Smad-binding in some nucleoporins
(Massague and Chen, 2000
;
Wrana, 2000
;
Moustakas et al., 2001
;
Randall et al., 2002
;
Xu et al., 2002
).
Role of XMAN1 in early embryogenesis
Interference with endogenous XMAN1 functions by morpholino oligos produced
defects in anterior development (Fig.
8). The affected regions, eyes and anterior CNS, correspond well
to the expression domains of XMAN1 at the neurula to tailbud stages (Figs
3,
8). Upregulation of the
epidermalizing activity of BMP signaling in XMAN1 morphants seems to be
responsible for the XMAN1-MO phenotype. The expression domain of Dlx3, an
epidermal marker, was expanded in XMAN1 morphants, and concomitantly, the
future neural plate was shortened and narrowed, particularly in the anterior
region where XMAN1 is normally expressed at the neurula stage. By contrast, in
XMAN1-MO-injected embryos, the expression of Sox2, a downstream target of
chordin and one of the earliest markers of the neuroectoderm
(Mizuseki et al., 1998a), was
not greatly affected at the gastrula stage (not shown). Because XMAN1 induces
neural tissues in animal caps independent of Chordin
(Fig. 6B), and dorsal
injections of XMAN1-MO do not affect the expression of chordin (not
shown), Sox2 expression may be regulated in an XMAN1-independent manner, or by
maternal XMAN1. It has been shown that translation from maternal mRNAs is not
efficiently inhibited by antisense morpholinos
(Heasman et al., 2000
).
Considering the fact that XMAN1 is initially expressed in the entire
ectoderm prior to gastrulation (Fig.
3B), maternal XMAN1 may sensitize the ectoderm to neural induction
by attenuating BMP signaling prior to organizer-derived BMP antagonists
(Fig. 6C).
Augmentation of BMP signaling by XMAN1-MO would affect other BMP-dependent
biological processes. We found that the development of XMAN1 morphants into
which XMAN1-MO was injected radially into the animal region was slower than
that of wild type after tailbud stages. Their development finally ceased and
they began to die. Because the TAK1-TAB1 pathway is activated in a
BMP-dependent manner and is involved in cell death
(Shibuya et al., 1998), it
should be interesting to investigate whether XMAN1 regulates the cell death
initiated by BMP the signaling.
Recently, it has been shown that the epithelial and sensorial layers of the
ectoderm have different competences to neuronal-promoting factors, and these
competences are established before gastrulation
(Chalmers et al., 2002). In
this sense, it should be noted that XMAN1 is expressed mainly in the sensorial
layer of the ectoderm at late blastula to neurula stages, where primary
neurons are generated. XMAN1 may be involved in generating the difference
between the two layers of neuroectoderm, perhaps establishing competence to
neurogenesis by modulating BMP signaling.
In this paper, we have presented the first evidence that an inner nuclear membrane protein is likely to have a role in signal transduction pathways. It will be interesting to analyze how XMAN1 functions as a modulator in BMP signaling during embryogenesis as well as in adulthood, when an autoimmune disease in which one of the autoantibody-reacting epitopes is MAN1 is known to occur in humans.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bateman, A., Birney, E., Cerruti, L., Durbin, R., Etwiller, L.,
Eddy, S. R., Griffiths-Jones, S., Howe, K. L., Marshall, M. and Sonnhammer, E.
L. (2002). The Pfam protein families database.
Nucleic Acids Res. 30,276
-280.
Bellefroid, E. J., Kobbe, A., Gruss, P., Pieler, T., Gurdon, J.
B. and Papalopulu, N. (1998). Xiro3 encodes a
Xenopus homolog of the Drosophila Iroquois genes and functions in
neural specification. EMBO J.
17,191
-203.
Birney, E., Kumar, S. and Krainer, A. R. (1993). Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res. 21,5803 -5816.[Abstract]
Casellas, R. and Brivanlou, A. H. (1998). Xenopus Smad7 inhibits both the activin and BMP pathways and acts as a neural inducer. Dev. Biol. 198, 1-12.[CrossRef][Medline]
Chalmers, A. D., Welchman, D. and Papalopulu, N. (2002). Intrinsic differences between the superficial and deep layers of the Xenopus ectoderm control primary neuronal differentiation. Dev. Cell 2, 171-182.[Medline]
Cohen, M., Lee, K. K., Wilson, K. L. and Gruenbaum, Y. (2001). Transcriptional repression, apoptosis, human disease and the functional evolution of the nuclear lamina. Trends Biochem. Sci. 26,41 -47.[CrossRef][Medline]
Dong, C., Li, Z., Alvarez, R., Jr, Feng, X. H. and Goldschmidt-Clermont, P. J. (2000). Microtubule binding to Smads may regulate TGFß activity. Mol. Cell 5, 27-34.[Medline]
Dye, B. T. and Patton, J. G. (2001). An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles. Exp. Cell Res. 263,131 -144.[CrossRef][Medline]
Eisaki, A., Kuroda, H., Fukui, A. and Asashima, M. (2000). XSIP1, a member of two-handed zinc finger proteins, induced anterior neural markers in Xenopus laevis animal cap. Biochem. Biophys. Res. Commun. 271,151 -157.[CrossRef][Medline]
Fainsod, A., Deissler, K., Yelin, R., Marom, K., Epstein, M., Pillemer, G., Steinbeisser, H. and Blum, M. (1997). The dorsalizing and neural inducing gene follistatin is an antagonist of BMP-4. Mech. Dev. 63,39 -50.[CrossRef][Medline]
Feledy, J. A., Beanan, M. J., Sandoval, J. J., Goodrich, J. S., Lim, J. H., Matsuo-Takasaki, M., Sato, S. M. and Sargent, T. D. (1999). Inhibitory patterning of the anterior neural plate in Xenopus by homeodomain factors Dlx3 and Msx1. Dev. Biol. 212,455 -464.[CrossRef][Medline]
Furukawa, K. (1999). LAP2 binding protein 1
(L2BP1/BAF) is a candidate mediator of LAP2-chromatin interaction.
J. Cell. Sci. 112,2485
-2492.
Grammer, T. C., Liu, K. J., Mariani, F. V. and Harland, R. M. (2000). Use of large-scale expression cloning screens in the Xenopus laevis tadpole to identify gene function. Dev. Biol. 228,197 -210.[CrossRef][Medline]
Harland, R. (2000). Neural induction. Curr. Opin. Genet. Dev. 10,357 -362.[CrossRef][Medline]
Harland, R. and Gerhart, J. (1997). Formation and function of Spemann's organizer. Annu. Rev. Cell. Dev. Biol. 13,611 -667.[CrossRef][Medline]
Harland, R. M. (1991). In situ hybridization: an improved whole-mount method for Xenopus embryos. In Methods in Cell Biology (ed. B. K. Kay and H. B. Peng), pp. 685-695. San Diego, CA: Academic Press.
Hata, A., Lagna, G., Massagué, J. and Hemmati-Brivanlou,
A. (1998). Smad6 inhibits BMP/Smad1 signaling by specifically
competing with the Smad4 tumor suppressor. Genes Dev.
12,186
-197.
Hata, A., Seoane, J., Lagna, G., Montalvo, E., Hemmati-Brivanlou, A. and Massague, J. (2000). OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways. Cell 100,229 -240.[Medline]
Heasman, J., Kofron, M. and Wylie, C. (2000). ß-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222,124 -134.[CrossRef][Medline]
Heldin, C. H., Miyazono, K. and ten Dijke, P. (1997). TGF-ß signalling from cell membrane to nucleus through SMAD proteins. Nature 390,465 -471.[CrossRef][Medline]
Hemmati-Brivanlou, A. and Thomsen, G. H. (1995). Ventral mesodermal patterning in Xenopus embryos: expression patterns and activities of BMP-2 and BMP-4. Dev. Genet. 17,78 -89.[Medline]
Hiratani, I., Mochizuki, T., Tochimoto, N. and Taira, M. (2001). Functional domains of the LIM homeodomain protein Xlim-1 involved in negative regulation, transactivation, and axis formation in Xenopus embryos. Dev. Biol. 229,456 -467.[CrossRef][Medline]
Hsu, D. R., Economides, A. N., Wang, X., Eimon, P. M. and Harland, R. M. (1998). The Xenopus dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities. Mol. Cell 1, 673-683.[Medline]
Iemura, S., Yamamoto, T. S., Takagi, C., Uchiyama, H., Natsume,
T., Shimasaki, S., Sugino, H. and Ueno, N. (1998). Direct
binding of follistatin to a complex of bone-morphogenetic protein and its
receptor inhibits ventral and epidermal cell fates in early Xenopus
embryo. Proc. Natl. Acad. Sci. USA
95,9337
-9342.
Inman, G., Nicolas, F. J. and Hill, C. S. (2002). Nucleoplasmic shuttling of Smads 2, 3, and 4 permits sensing og TGF-ß receptor activity. Mol. Cell 10,283 -294.[Medline]
Kay, B. K. and Peng, H. B. (1991). Xenopus laevis: Practical Uses in Cell and Molecular Biology. San Diego, CA: Academic Press.
Knecht, A. K., Good, P. J., Dawid, I. B. and Harland, R. M.
(1995). Dorsal-ventral patterning and differentiation of
noggin-induced neural tissue in the absence of mesoderm.
Development 121,1927
-1935.
Kroll, K. L., Salic, A. N., Evans, L. M. and Kirschner, M.
W. (1998). Geminin, a neuralizing molecule that demarcates
the future neural plate at the onset of gastrulation.
Development 125,3247
-3258.
Lee, K. K., Haraguchi, T., Lee, R. S., Koujin, T., Hiraoka, Y. and Wilson, K. L. (2001). Distinct functional domains in emerin bind lamin A and DNA-bridging protein BAF. J. Cell Sci. 114,4567 -4573.[Medline]
Lin, F., Blake, D. L., Callebaut, I., Skerjanc, I. S., Holmer,
L., McBurney, M. W., Paulin-Levasseur, M. and Worman, H. J.
(2000). MAN1, an inner nuclear membrane protein that shares the
LEM domain with lamina-associated polypeptide 2 and emerin. J.
Biol. Chem. 275,4840
-4847.
Mangold, O. and Spemann, H. (1927). Über induction von Medullarplatte durch Medullarplatte im jüngeren Keim, ein Beispiel homöogenetischer oder assimilatorischer Induction. Wilhelm Roux' Arch. Entwicklungsmech. Org. 111,341 -422.
Manilal, S., Nguyen, T. M., Sewry, C. A. and Morris, G. E.
(1996). The Emery-Dreifuss muscular dystrophy protein, emerin, is
a nuclear membrane protein. Hum. Mol. Genet.
5, 801-808.
Mariani, F. V. and Harland, R. M. (1998). XBF-2
is a transcriptional repressor that converts ectoderm into neural tissue.
Development 125,5019
-5031.
Massagué, J. and Chen, Y. G. (2000).
Controlling TGF-ß signaling. Genes Dev.
14,627
-644.
Mathers, P. H., Grinberg, A., Mahon, K. A. and Jamrich, M. (1997). The Rx homeobox gene is essential for vertebrate eye development. Nature 387,603 -607.[CrossRef][Medline]
Mizuseki, K., Kishi, M., Matsui, M., Nakanishi, S. and Sasai,
Y. (1998a). Xenopus Zic-related-1 and Sox-2, two
factors induced by chordin, have distinct activities in the initiation of
neural induction. Development
125,579
-587.
Mizuseki, K., Kishi, M., Shiota, K., Nakanishi, S. and Sasai, Y. (1998b). SoxD: an essential mediator of induction of anterior neural tissues in Xenopus embryos. Neuron 21,77 -85.[Medline]
Moustakas, A., Souchelnytskyi, S. and Heldin, C. H. (2001). Smad regulation in TGF-ß signal transduction. J. Cell. Sci. 114,4359 -4369.[Medline]
Nagano, A., Koga, R., Ogawa, M., Kurano, Y., Kawada, J., Okada, R., Hayashi, Y. K., Tsukahara, T. and Arahata, K. (1996). Emerin deficiency at the nuclear membrane in patients with Emery-Dreifuss muscular dystrophy. Nat. Genet. 12,254 -259.[Medline]
Nakata, K., Nagai, T., Aruga, J. and Mikoshiba, K.
(1997). Xenopus Zic3, a primary regulator both in neural
and neural crest development. Proc. Natl. Acad. Sci.
USA 94,11980
-11985.
Nakayama, T., Gardner, H., Berg, L. K. and Christian, J. L.
(1998). Smad6 functions as an intracellular antagonist of some
TGF-ß family members during Xenopus embryogenesis.
Genes Cells 3,387
-394.
Nieuwkoop, P. D. and Faber, J. (1967).Normal Table of Xenopus laevis (Daudin) . Amsterdam: North Holland.
Onichtchouk, D., Chen, Y. G., Dosch, R., Gawantka, V., Delius, H., Massague, J. and Niehrs, C. (1999). Silencing of TGF-ß signalling by the pseudoreceptor BAMBI. Nature 401,480 -485.[CrossRef][Medline]
Osada, S. I. and Wright, C. V. (1999).
Xenopus nodal-related signaling is essential for mesendodermal
patterning during early embryogenesis. Development
126,3229
-3240.
Pannese, M., Lupo, G., Kablar, B., Boncinelli, E., Barsacchi, G. and Vignali, R. (1998). The Xenopus Emx genes identify presumptive dorsal telencephalon and are induced by head organizer signals. Mech. Dev. 73,73 -83.[CrossRef][Medline]
Paulin-Levasseur, M., Blake, D. L., Julien, M. and Rouleau, L. (1996). The MAN antigens are non-lamin constituents of the nuclear lamina in vertebrate cells. Chromosoma 104,367 -379.[CrossRef][Medline]
Penzel, R., Oschwald, R., Chen, Y., Tacke, L. and Grunz, H. (1997). Characterization and early embryonic expression of a neural specific transcription factor xSOX3 in Xenopus laevis. Int. J. Dev. Biol. 41,667 -677.[Medline]
Piccolo, S., Agius, E., Leyns, L., Bhattacharyya, S., Grunz, H., Bouwmeester, T. and De Robertis, E. M. (1999). The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. Nature 397,707 -710.[CrossRef][Medline]
Randall, R. A., Germain, S., Inman, G. J., Bates, P. A. and
Hill, C. S. (2002). Different Smad2 partners bind a common
hydrophobic pocket in Smad2 via a defined proline-rich motif. EMBO
J. 21,145
-156.
Sasai, Y., Lu, B., Steinbeisser, H. and De Robertis, E. M. (1995). Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus. Nature 376,333 -336.[CrossRef][Medline]
Sasaki, A., Masuda, Y., Ohta, Y., Ikeda, K. and Watanabe, K.
(2001). Filamin associates with Smads and regulates transforming
growth factor-ß signaling. J. Biol. Chem.
276,17871
-17877.
Segura-Totten, M., Kowalski, A. K., Craigie, R. and Wilson, K.
L. (2002). Barrier-to-autointegration factor: major roles in
chromatin decondensation and nuclear assembly. J. Cell
Biol. 158,475
-485.
Servetnick, M. and Grainger, R. M. (1991). Homeogenetic neural induction in Xenopus. Dev. Biol. 147, 73-82.[Medline]
Shibata, M., Itoh, M., Ohmori, S.-y., Shinga, J. and Taira, M. (2001). Systematic screening and expression analysis of the head organizer genes in Xenopus embryos. Dev. Biol. 239,241 -256.[CrossRef][Medline]
Shibuya, H., Iwata, H., Masuyama, N., Gotoh, Y., Yamaguchi, K.,
Irie, K., Matsumoto, K., Nishida, E. and Ueno, N. (1998).
Role of TAK1 and TAB1 in BMP signaling in early Xenopus development.
EMBO J. 17,1019
-1028.
Shumaker, D. K., Lee, K. K., Tanhehco, Y. C., Craigie, R. and
Wilson, K. L. (2001). LAP2 binds to BAF.DNA complexes:
requirement for the LEM domain and modulation by variable regions.
EMBO J. 20,1754
-1764.
Souchelnytskyi, S., Nakayama, T., Nakao, A., Moren, A., Heldin,
C. H., Christian, J. L. and ten Dijke, P. (1998). Physical
and functional interaction of murine and Xenopus Smad7 with bone
morphogenetic protein receptors and transforming growth factor-ß
receptors. J. Biol. Chem.
273,25364
-25370.
Suzuki, A., Ueno, N. and Hemmati-Brivanlou, A.
(1997). Xenopus msx1 mediates epidermal induction and neural
inhibition by BMP4. Development
124,3037
-3044.
Taira, M., Otani, H., Saint-Jeannet, J. P. and Dawid, I. B. (1994). Role of the LIM class homeodomain protein Xlim-1 in neural and muscle induction by the Spemann organizer in Xenopus.Nature 372,677 -679.[CrossRef][Medline]
Tsukazaki, T., Chiang, T. A., Davison, A. F., Attisano, L. and Wrana, J. L. (1998). SARA, a FYVE domain protein that recruits Smad2 to the TGFß receptor. Cell 95,779 -791.[Medline]
Verschueren, K., Remacle, J. E., Collart, C., Kraft, H., Baker,
B. S., Tylzanowski, P., Nelles, L., Wuytens, G., Su, M. T., Bodmer, R., Smith,
J. C. and Huylebroeck, D. (1999). SIP1, a novel zinc
finger/homeodomain repressor, interacts with Smad proteins and binds to
5'-CACCT sequences in candidate target genes. J. Biol.
Chem. 274,20489
-20498.
von Bubnoff, A. and Cho, K. W. (2001). Intracellular BMP signaling regulation in vertebrates: pathway or network? Dev. Biol. 239,1 -14.[CrossRef][Medline]
Wang, W., Mariani, F. V., Harland, R. M. and Luo, K.
(2000). Ski represses bone morphogenic protein signaling in
Xenopus and mammalian cells. Proc. Natl. Acad. Sci.
USA 97,14394
-14399.
Whitman, M. (1998). Smads and early
developmental signaling by the TGFß superfamily. Genes
Dev. 12,2445
-2462.
Worman, H. J. and Courvalin, J. C. (2000). The inner nuclear membrane. J. Membr. Biol. 177, 1-11.[CrossRef][Medline]
Wrana, J. L. (2000). Regulation of Smad activity. Cell 100,189 -192.[Medline]
Wu, W., Lin, F. and Worman, H. J. (2002).
Intracellular trafficking of MAN1, an integral protein of the nuclear envelope
inner membrane. J. Cell. Sci.
115,1361
-1371.
Xiao, Z., Watson, N., Rodriguez, C. and Lodish, H. F.
(2001). Nucleocytoplasmic shuttling of Smad1 conferred by its
nuclear localization and nuclear export signals. J. Biol.
Chem. 276,39404
-39410.
Xu, L., Kang, Y., Col, S. and Massagué, J. (2002). Smad2 nucleocytoplasmic shuttling by nucleoporins CAN/Nup214 and Nup153 feeds TGFß signaling complexes in the cytoplasm and nucleus. Mol. Cell 10,271 -282.[Medline]
Zhang, Y., Chang, C., Gehling, D. J., Hemmati-Brivanlou, A. and
Derynck, R. (2001). Regulation of Smad degradation and
activity by Smurf2, an E3 ubiquitin ligase. Proc. Natl. Acad. Sci.
USA 98,974
-979.
Zhou, X., Hollemann, T., Pieler, T. and Gruss, P. (2000). Cloning and expression of xSix3, the Xenopus homologue of murine Six3. Mech. Dev. 91,327 -330.[CrossRef][Medline]
Zhu, H., Kavsak, P., Abdollah, S., Wrana, J. L. and Thomsen, G. H. (1999). A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature 400,687 -693.[CrossRef][Medline]
Zimmerman, L. B., De Jesus-Escobar, J. M. and Harland, R. M. (1996). The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86,599 -606.[Medline]
Related articles in Development: