1 Department of Biological Sciences, Graduate School of Bioscience and
Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama
226-8501, Japan
2 Department of Biological Sciences, Graduate School of Science, Tokyo
Metropolitan University, Minamiohsawa, Hachiohji, Tokyo 192-0397, Japan
Author for correspondence (e-mail:
kobaken{at}ascidian.zool.kyoto-u.ac.jp)
Accepted 14 July 2003
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SUMMARY |
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Key words: Mesenchyme, Notochord, Induction, Responsiveness, Intrinsic factor, macho-1, FGF, snail, Ascidian
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Introduction |
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The structure of the ascidian tadpole larva is relatively simple. As shown
in Fig. 1A, larval muscle cells
lie laterally on both sides of the notochord, which is aligned in the center
of the tail. The posterior nerve cord is located on the dorsal side of the
notochord in the tail. On each side of the trunk region, there is a cluster of
mesenchyme cells. Fig. 1B,C
show fate maps of the vegetal hemisphere at the blastula stage (32- and
64-cell stage in ascidians) (Nishida,
1987; Kim et al.,
2000
; Minokawa et al.,
2001
). Nerve cord, notochord, mesenchyme and muscle cells are
derived from the anterior and posterior margins of the vegetal hemisphere. The
endoderm originates from the central zone. These five tissue-forming areas are
aligned along the anteroposterior axis. From the anterior, nerve cord,
notochord, endoderm, mesenchyme and muscle precursors are present in this
order.
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Thus, there are striking similarities at the cellular and molecular levels
between mesenchyme and notochord induction, and a similar mechanism
symmetrically functions in both the anterior and posterior marginal zones. In
particular, the signaling cascade from FGF to MAPK (ERK1/2) is remarkably
conserved among mesenchyme and notochord induction in ascidian embryos, as
well as in other organisms that have been studied. But mesenchyme and
notochord blastomeres show distinct responses to the same FGF signal. Removal
and transplantation of egg cytoplasm by microsurgery revealed that the
difference in their responsiveness is caused by the cytoplasmic factor of the
responding blastomeres, which is inherited from the egg
(Kim et al., 2000)
(Fig. 1E,F). The
posterior-vegetal cytoplasm (PVC) of eggs confers the muscle and mesenchyme
fate on the posterior blastomeres. Removal of the PVC resulted in
anteriorization of the embryos. Blastomeres positioned where mesenchyme
blastomeres are normally located were converted to notochord, so that central
endoderm blastomeres were encircled by notochord blastomeres. Thus, removal of
the PVC causes ectopic formation of notochord and loss of mesenchyme in the
posterior region (Fig. 1E).
However, transplantation of the PVC to the anterior region of another intact
egg suppressed notochord formation and promoted ectopic formation of
mesenchyme in the anterior blastomeres
(Fig. 1F). Therefore, the
factors that are localized in the PVC are involved in differentiating cell
response to the FGF signal. In the presence of the PVC factors, blastomeres
respond by forming mesenchyme; and, in their absence, blastomeres respond by
developing into notochord.
The molecular nature of the PVC factors that determine cellular
responsiveness is unknown. The PVC is the region corresponding to Conklin's
myoplasm at completion of ooplasmic segregation
(Conklin, 1905). Recently, it
was shown that maternal mRNA of macho-1, which is localized in the
PVC region, is an ascidian muscle determinant
(Nishida and Sawada, 2001
).
macho-1 encodes a putative transcription factor with zinc-finger
domains. macho-1 would be a good candidate for the PVC factor that
regulates cellular responsiveness. Other evidence to support this idea is that
without induction, mesenchyme blastomeres assume muscle fate directed by
macho-1 (Kim and Nishida,
1999
). Therefore, macho-1 products are supposed to be
present also in mesenchyme blastomeres, and can play a role as the PVC factor.
To examine this hypothesis, we investigated the formation of mesenchyme,
notochord, muscle and nerve cord in macho-1-deficient and
macho-1-overexpressing embryos. Our results showed that
macho-1 not only is a muscle determinant but also plays a pivotal
role as an intrinsic factor that controls the responsiveness of mesenchyme
blastomeres.
Downstream of the maternal PVC factor, zygotic events would be involved in
suppression of the notochord fate in mesenchyme blastomeres. snail is
a possible candidate, because it is expressed in muscle and mesenchyme
precursors at the 32-cell stage or the 44-cell stage
(Erives et al., 1998;
Wada and Saiga, 1999
).
Furthermore, Snail is a zinc-finger protein known to be a transcription
repressor. Brachyury is a key transcription factor that is involved
in notochord formation in ascidians (Yasuo
and Satoh, 1998
; Takahashi et
al., 1999a
). Misexpression of snail in notochord-lineage
cells driven by a heterologous promoter suppresses at least the expression of
the reporter gene driven by the Brachyury minimal promoter through
Snail-binding sites within it, although the formation of notochord was not
suppressed in experiments (Fujiwara et al.,
1998
). We showed that snail is a downstream target of
maternal macho-1. To examine the function of snail in
mesenchyme and notochord induction, we also injected snail mRNA into
eggs, where it suppressed endogenous Brachyury expression and
formation of notochord.
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Materials and methods |
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Injection of MO and synthetic mRNAs
To suppress translation of macho-1, we used antisense morpholino
oligonucleotides (MO; Gene Tools) complementary to the 5' UTR of
macho-1 (GenBank Accession Number: AB045124)
(5'-AATTGCAAAACACAAAAATCACACG-3', antisense to a 25-nucleotide
sequence spanning nucleotides 13-37 of macho-1 cDNA). In control
experiments, we used 4-mismatch control MO
(5'-AATTCCAAATCACAATAATCTCACG-3',
mismatch underlined). Capped mRNAs of macho-1 and Hrsna were
synthesized as described previously
(Nishida and Sawada, 2001),
except for the use of the mMessage mMachine kit (Ambion). Mutant
macho-1 mRNA lacking a zinc-finger domain
(Nishida and Sawada, 2001
) and
lacZ mRNA were used as controls. MO and/or synthetic mRNA was
suspended in sterile distilled water and injected into intact eggs after
fertilization. For microinjection, we followed the method described previously
(Miya et al., 1997
).
Isolation of blastomeres and inhibition of cell division
Embryos were manually devitellinated with tungsten needles and reared in
1.2% agar-coated plastic dishes filled with seawater. Blastomeres were
identified and isolated from embryos with a fine glass needle under a
stereomicroscope (SZX-12; Olympus). Isolated blastomeres were cultured
separately as partial embryos in agar-coated plastic dishes, then the partial
embryos were fixed for immunohistochemistry or in situ hybridization. To
inhibit cell division, cleavage was permanently arrested with 2.5 µg/ml
cytochalasin B (Sigma) at the 110-cell stage.
Removal and transplantation of egg cytoplasm
Removal and transplantation of PVC were carried out as described previously
(Nishida, 1994). After
completion of ooplasmic segregation, fertilized eggs were oriented by using
the position of the polar bodies and the posterior transparent myoplasm. Egg
fragments containing the PVC, which was 8%-15% of the total egg volume, were
removed from the eggs by severing the eggs with a fine glass needle. The eggs
were cultured as PVC-deficient embryos. For transplantation of the PVC, an egg
fragment containing PVC that had been severed from an egg was transplanted
into the anterior-vegetal region of another intact egg by using polyethylene
glycol and electric field-mediated fusion.
Treatment with FGF
Isolated blastomeres were transferred into seawater that contained 0.1%
bovine serum albumin (BSA; Sigma) and 2 ng/ml recombinant human bFGF protein
(Amersham). This concentration of FGF is effective enough to induce notochord
and mesenchyme formation in Halocynthia
(Nakatani et al., 1996;
Kim et al., 2000
). In
controls, blastomeres were treated with BSA in seawater.
Immunohistochemistry
Formation of mesenchyme cells was monitored by staining with the Mch-3
monoclonal antibody (Kim and Nishida,
1998). The monoclonal antibody Mu-2 was used for monitoring muscle
formation (Nishikata et al.,
1987
). It recognizes the myosin heavy chain of
Halocynthia (Makabe and Satoh,
1989
). The specimens were fixed after the hatching stage for 10
minutes in methanol at -20°C. The monoclonal antibody Not1 recognized a
component of the notochordal sheath that is secreted by notochord cells
(Nishikata and Satoh, 1990
).
At the middle tailbud stage, this antibody is strictly specific to notochord
cells (Nakatani and Nishida,
1994
). Specimens were fixed at the middle tailbud stage for Not1
staining. Indirect immunofluorescence detection was carried out by standard
methods using a TSA fluorescein system (PerkinElmer Life Sciences) according
to the manufacturer's protocol.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was carried out as described previously
(Wada et al., 1995). Specimens
were hybridized by using digoxigenin-labeled HrBra, HrETR-1 and
Hrsna antisense probes. HrBra, encoding Halocynthia
Brachyury gene, was used to assess notochord specification
(Yasuo and Satoh, 1993
). The
expression of HrBra was monitored at the 110-cell stage.
HrETR-1, encoding an RNA-binding protein of the Elav family, was used
as a molecular marker for nerve cord specification
(Yagi and Makabe, 2001
). The
expression of HrETR-1 was monitored at the 118-cell stage or the
neural-plate stage in cleavage-arrested 110-cell embryos. Hrsna
encodes a Snail homolog in Halocynthia
(Wada and Saiga, 1999
). The
expression was examined at the 64-cell stage.
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Results |
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In control larvae derived from fertilized eggs injected with 4-mismatch
control MO, mesenchyme cell clusters were normally detected by immunostaining
of mesenchyme-specific Mch3 antigen in 20 cases
(Fig. 3A). By contrast,
injection of 100 pg of macho-1 MO abolished the expression of the
antigen in all 13 cases (Fig.
3B). This was also confirmed in another way. In ascidian embryos,
even when cleavages were permanently arrested with cytochalasin B at a
cleavage stage, cleavage-arrested blastomeres continued some differentiation
processes and eventually expressed different features according to their
developmental fates (Whittaker,
1973; Nishikata and Satoh,
1990
). In embryos injected with control MO, four mesenchyme
precursors (B8.5 and B7.7 blastomere pairs) eventually expressed the
mesenchyme marker, as expected from the cell lineage when cleavage and
morphogenesis were arrested at the 110-cell stage
(Fig. 3C,D) (four cells were
stained in 74% of 15 cases). Expression of the mesenchyme marker was also
suppressed by macho-1 MO injection in cleavage-arrested 110-cell
embryos (Fig. 3E) (four cells
were stained in 8% of 13 cases). This phenotype coincides well with the
PVC-deficient embryos (Fig. 3F) (Kim et al., 2000
). As will be
described later, mesenchyme precursors in macho-1-deficient embryos
assumed a notochord fate.
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To directly confirm the fate conversion of mesenchyme to notochord, presumptive mesenchyme blastomeres (B8.5 and B7.7 in Fig. 4I) were identified and isolated at the 110-cell stage and cultured as partial embryos without cleavage-arrest. Expression of Not1 was never observed in B8.5 (n=21) or B7.7 (n=18) partial embryos injected with control MO (Fig. 4J,L). By contrast, B8.5 and B7.7 partial embryos isolated from embryos injected with macho-1 MO (100 pg) expressed Not1 in 63% (n=40) and 39% (n=53) of cases, respectively (Fig. 4K,M).
Transplantation of the PVC to the anterior region suppressed notochord formation (Fig. 1F). To examine whether macho-1 overexpression reproduces the phenotype of PVC-transplanted embryos, we examined notochord formation in macho-1-overexpressing embryos. Injection of macho-1 mRNA resulted in the decrease (50 pg, n=32) or loss (100 pg, n=29) of Not1 expression in cleavage-arrested 110-cell embryos and HrBra expression at the 110-cell stage (Fig. 4D,H,H'). In these cases, as mentioned in the previous section, presumptive notochord blastomeres presumably failed to develop into notochord because they assumed mesenchyme or muscle fates.
Muscle precursor blastomeres assume nerve cord fate without
macho-1
macho-1-deficient embryos lose primary muscle cells
(Fig. 2B,C)
(Nishida and Sawada, 2001).
However, it is not known what kind of tissue cell the muscle precursor cells
are converted to in those embryos, although we expected it to be nerve cord
(Fig. 1E). Formation of nerve
cord has not yet been examined in PVC-removed and -transplanted embryos or in
macho-1-deficient and -overexpressing embryos. Therefore, we examined
muscle and nerve cord formation in these embryos to fully understand fate
specification in the marginal zone of early ascidian embryos, although this
issue is not directly relevant to the mechanisms that control cellular
responsiveness.
Fig. 5J shows a fate map of
110-cell stage embryos; 10 presumptive primary-muscle blastomeres are colored
red. First, we examined muscle formation in macho-1-deficient and
-overexpressing embryos. In control embryos, when cleavage was arrested at the
110-cell stage, 10 muscle precursors eventually expressed muscle myosin, as
expected from the fate map (Fig.
5A). In embryos injected with macho-1 MO, expression of
muscle myosin was suppressed (Fig.
5C), as in the PVC-deficient embryos
(Fig. 5B). By contrast,
overexpression of the macho-1 mRNA resulted in ectopic formation of
muscle cells (Fig. 5D), as
observed previously in PVC-transplanted embryos
(Nishida, 1994). All of these
results reconfirm the previous results, showing the validity of
macho-1 MO and mRNA, as well as cytoplasmic removal and
transplantation in the present study.
Eight nerve cord precursor blastomeres are colored purple in the fate map
(Fig. 5J). We investigated
nerve cord formation by monitoring the expression of a neural plate marker
gene, HrETR-1, in PVC-removed embryos and macho-1-deficient
embryos. The expression of HrETR-1 is restricted in neural plate
precursors at the 110- or 118-cell stage in ascidian embryos
(Fig. 5E,E')
(Yagi and Makabe, 2001).
HrETR-1 gene expression was monitored in cleavage-arrested 110-cell
embryos and 118-cell embryos without cleavage-arrest. In cleavage-arrested
110-cell embryos (n=30) and 118-cell embryos (n=33) injected
with control MO, nerve cord precursors expressed HrETR-1, as expected
from fate map (Fig.
5E,E'). The number of HrETR-1-positive blastomeres
in the vegetal hemisphere ranged form six to eight.
Removal of the PVC (total number examined=60) resulted in ectopic expression of HrETR-1 in the entire marginal zone (Fig. 5F,F'). Thus, presumptive muscle blastomeres assumed nerve cord fate in these embryos. In embryos injected with macho-1 MO (total number examined=87), ectopic expression of HrETR-1 was also observed in the lateral and posterior region, which corresponds to muscle blastomeres (Fig. 5G,G', white arrowhead). Ectopic nerve cord formation in the posterior-vegetal region in macho-1-deficient embryos was also confirmed by isolation of blastomeres at the eight-cell stage (data not shown). The nerve cord phenotype was similar in both macho-1-deficient and PVC-deficient embryos. However, in macho-1-deficient embryos, the posterior (B7.5) cells never expressed HrETR-1 (Fig. 5G', black arrowheads), while in PVC removed embryos, the posterior cells were ectopically expressed HrETR-1. We noticed that the posterior (B7.5) blastomeres in macho-1-deficient embryos assumed an endoderm fate but not a nerve cord fate (data not shown). We then examined nerve cord formation in macho-1-overexpressing embryos and PVC-transplanted embryos. In embryos injected with macho-1 mRNA (100 pg), HrETR-1 expression was completely suppressed (n=34) (Fig. 5H), as well as in the PVC-transplanted embryos (n=8) (Fig. 5I). These results indicate that muscle precursors assume a nerve cord fate without macho-1.
Zygotic expression of snail is downstream of
macho-1 and inhibits notochord formation
snail seems a good candidate for mediating suppression of a
notochord fate in presumptive mesenchyme precursors. We first examined whether
zygotic expression of a snail homolog, Hrsna, occurs
downstream of maternal macho-1
(Fig. 6A). Injection of control
mutant form of macho-1 mRNA (100 pg) had no effect on Hrsna
expression at the 64-cell stage (n=32). When macho-1 mRNA
(100 pg) was injected, Hrsna expression was ectopically activated
(n=39). However, injection of macho-1 MO (100 pg) suppressed
Hrsna expression (n=22). Therefore, macho-1 is
necessary and sufficient for Hrsna expression.
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Discussion |
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The effect of macho-1 MO
We used antisense morpholino oligonucleotides (MO) to prevent the function
of macho-1 by inhibiting its translation. The phenotype caused by a
low dose injection (100 pg) is almost the same as that seen in our previous
study using phosphorothioate DNA oligonucleotides (S-DNA) to deplete
macho-1 mRNA (Nishida and Sawada,
2001). However, a high dose injection (300 pg) resulted in a
phenotype that is similar to that of PVC-removed larvae. This phenotype is
unlikely to have been the result of non-specific toxic effects, because
notochord cells were formed in every macho-1-deficient embryo, and
embryonic cells transfated rather than failed to differentiate. Thus, it is
plausible that MO inhibited the macho-1 functions more efficiently
than did S-DNA. This conclusion is supported by the observation that injection
of another MO that covers a different region of macho-1 mRNA produced
similar phenotypes as the main MO we used in this studies (data not
shown).
No muscle cells formed in high-dose-injected embryos
(Fig. 2C). This phenotype could
be an indirect effect of the prevention of macho-1 functions.
Formation of the primary lineage (B-line) of muscle cells depends on maternal
macho-1 (Nishida and Sawada,
2001). However, the fate of the secondary lineage (A-line and
b-line) of muscle cells is specified by cell interactions, probably during
gastrulation (Nishida, 1990
).
Although the inducer cells and the inducing signal involved in secondary
muscle formation are as yet unknown, it is possible that inhibition of the
macho-1 function would perturb secondary muscle induction.
macho-1 is not only a muscle determinant but also a main
component of the PVC factor
We investigated the formation of mesenchyme and notochord in
macho-1-deficient and -overexpressing embryos. In
macho-1-deficient embryos, mesenchyme formation was completely
suppressed, and instead ectopic notochord formation was promoted in the
presumptive mesenchyme precursors. Conversely, in
macho-1-overexpressing embryos, notochord formation was suppressed,
and ectopic mesenchyme formation was observed in the anterior-vegetal region.
These phenotypes were the same as those of PVC-removed embryos and
PVC-transplanted embryos, respectively. These results support the idea that
maternal mRNA of macho-1, first identified as a muscle determinant,
also plays a role as an intrinsic factor that controls the responsiveness of
mesenchyme blastomeres. Most importantly, in embryos injected with
macho-1 mRNA, treatment with FGF led to ectopic mesenchyme formation
even in animal blastomeres. This result provides strong evidence that
macho-1 plays a key role in determining that cells are induced to
develop into mesenchyme when they receive the FGF signal.
macho-1-deficient embryos reproduced only some of the phenotypes
of the PVC-removed embryos. In the PVC-removed embryos, in addition to loss of
muscle and mesenchyme, the cleavage pattern of the posterior-vegetal region
was converted to that of the anterior-vegetal region
(Nishida, 1994). The cleavage
pattern of macho-1-deficient embryos was normal at least up to the
gastrula stage. Therefore, PVC is likely to have two distinct functions. One
function is muscle and mesenchyme formation, and is accounted for by maternal
mRNA of macho-1. Another function of PVC is the generation of the
posterior cleavage pattern. It has been reported that a unique subcellular
structure designated the centrosome-attracting body (CAB), which exists in the
posterior pole cortex of cleaving embryos, plays essential roles in generating
the posterior cleavage pattern and the unequal cleavages within it
(Hibino et al., 1998
;
Nishikata et al., 1999
;
Iseto and Nishida, 1999
).
Removal of the PVC results in loss of the CAB. Transplantation of the PVC into
the anterior region causes ectopic formation of the CAB in the anterior
region, and the cleavage pattern of the anterior region converts to the
posterior type (Nishida, 1994
;
Nishikata et al., 1999
).
Therefore, another molecule involved in the formation of the CAB would also be
present in the PVC. Several kinds of maternal mRNA show a similar localization
pattern to that of macho-1, namely localization to PVC in fertilized
eggs (Yoshida et al., 1996
;
Satou and Satoh, 1997
;
Sasakura et al., 1998a
;
Sasakura et al., 1998b
;
Sasakura et al., 2000
;
Satou, 1999
;
Caracciolo et al., 2000
;
Makabe et al., 2001
;
Nishida and Sawada, 2001
;
Nishikata et al., 2001
;
Nakamura et al., 2003
).
Snail is downstream of macho-1 and mediates
suppression of notochord fate in mesenchyme precursors
Expression of ascidian snail preferentially starts in the
mesenchyme-muscle precursor blastomeres at the 32-cell stage or the 44-cell
stage (Erives et al., 1998;
Wada and Saiga, 1999
). Our
preliminary results show that the expression of snail depends on the
presence of the PVC (A. Yamada, H. Yamamoto and H. Nishida, unpublished).
Similarly, the phenotype of macho-1-deficient and -overexpressing
embryos indicates that macho-1 is necessary and sufficient for
zygotic Hrsna expression in these blastomeres. The overexpression of
Hrsna reduced HrBra and Not1 antigen expression. These
results suggest that zygotic Hrsna expression mediates the
suppression of notochord fate by maternal macho-1 in the posterior
region. This idea is supported by the following observations. Snail is a
zinc-finger protein known to be a transcription repressor in
Drosophila (Ip et al.,
1992
). In Ciona intestinalis, misexpression of
snail in notochord-lineage cells driven by a heterologous promoter
suppresses at least the expression of the reporter gene driven by the
Brachyury minimal promoter through Snail-binding sites within it.
However, neither endogenous Brachyury expression nor the formation of
notochord was suppressed in experiments
(Fujiwara et al., 1998
),
contrary to our results.
The difference between these previous results and ours is the mode of
misexpression. In Ciona, misexpression of snail was driven
by the Brachyury promoter, which promotes misexpression after the
64-cell stage, whereas snail expression starts at the 32-cell stage
or the 44-cell stage in normal embryos. That stage could be too late for
misexpressed snail to suppress initiation of endogenous
Brachyury expression. In the present study, we injected synthetic
snail mRNA into eggs. Therefore, enough protein could accumulate
before the initiation of Brachyury expression. Of course, the
difference may be attributed to species difference. But this explanation is
unlikely, because the promoters of Ciona and Halocynthia
Brachyury are interchangeable and are able to drive notochord expression
in either species (Takahashi et al.,
1999b). It is not known whether there is a Snail-binding site in
the Halocynthia Brachyury promoter, but above-mentioned observations
suggests that a similar mechanism operates in Ciona and
Halocynthia. Thus, our results confirm that snail is indeed
involved in suppression of notochord fate in the posterior blastomeres. To
confirm the role of Hrsna in suppression of notochord fate, we
injected MO complementary to Hrsna. However, there was no ectopic
notochord formation. At the moment, it is not clear whether the Hrsna
MO was not sufficiently effective or there is a redundant mechanism to
suppress notochord fate other than that involving Hrsna. Furthermore,
overexpression of Hrsna was not enough to promote ectopic mesenchyme
formation, suggesting that Hrsna is involved only in suppression of a
notochord fate but not in promotion of a mesenchyme fate.
Model for fate specification in the vegetal-marginal cells of
ascidian embryos: two-step model
The default fates of mesenchyme precursors and notochord precursors are
muscle and nerve cord, respectively (Fig.
1D). In this study, we also investigated formation of muscle and
nerve cord in macho-1-deficient and -overexpressing embryos. In
macho-1-deficient embryos, the formation of muscle was suppressed,
and HrETR-1, a neural plate marker gene, was ectopically expressed in
the presumptive muscle blastomeres. On the other hand, in
macho-1-overexpressing embryos, HrETR-1 expression was
suppressed and ectopic formation of muscle was observed. These results
together led us to propose a simple model for fate specification in ascidian
embryos (Fig. 7).
|
The future question is how cells integrate the intrinsic activity of
macho-1 with information from extrinsic cues that are delivered into
the cell by the signal-transduction machinery. The Macho-1 protein has five
CCHH-type zinc-finger repeats that show similarity with Zic, GLI and
odd-paired proteins (Nishida and Sawada,
2001). All of these proteins are transcription factors. Because
Macho-1 protein synthesized from FLAG-tagged mRNAs accumulates in the nuclei
during the cleavage stage, it was suggested that Macho-1 functions as a
transcription factor (Nishida and Sawada,
2001
). Our recent results using VP16 and EnR fusion
protein further support the possibility that Macho-1 indeed functions as a
transcription activator (K. Sawada and H. Nishida, unpublished). Recently, we
also found that an Ets transcription factor is the target activated by
FGF-MAPK (ERK1/2) signaling and is involved in notochord and mesenchyme
induction in ascidians (Miya and Nishida,
2003
). Thus, it is important to elucidate how these two
transcription factors cooperate to promote mesenchyme fate. There are two
possibilities. The Ets transcription factor is known to interact with other
transcription factors to direct signals for the transcription of specific
target genes (Sharrocks,
2001
). For example, it has been shown that mammalian Ets1
interacts with Pit-1, a pituitary-specific POU-homeodomain protein, and
activates the transcription of pituitary-specific genes
(Bradford et al., 1997
).
Another possibility is that inputs from the signal resulting in Ets activation
and Macho-1 activity could be combined at the level of regulatory regions of
the target genes without direct interaction of either Ets or Macho-1 protein.
Both factors might independently bind to cis-regulatory elements and
cooperate to activate or silence the target gene transcription.
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ACKNOWLEDGMENTS |
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Footnotes |
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