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First published online 14 March 2007
doi: 10.1242/dev.02825
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1 Faculty of Marine Bioscience and Technology, Kangnung National University,
Gangneung Daehangno, Gangneung 210-702, Republic of Korea.
2 Department of Biological Sciences, Graduate School of Science, Osaka
University, Toyonaka, Osaka 560-0043, Japan.
* Author for correspondence (e-mail: gjkim{at}kangnung.ac.kr)
Accepted 31 January 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Ascidian embryo, Mesenchyme, Notochord, Embryonic induction, Asymmetric cell division, FGF, FoxA
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Howkins and Garriga,
1998; Goldstein,
2000; Knoblich,
2001; Roegiers and Jan,
2004; Betschinger and Knoblich,
2004). How is the polarity of asymmetry determined and
established? From extensive studies using Caenorhabditis elegans and
Drosophila as model systems, asymmetric cell divisions are known to
involve both polarized intrinsic factors and inductive signals. As examples of
asymmetric cell divisions using external cues, the EMS cell of the C.
elegans four-cell-stage embryo and the T cell at a later postembryonic
stage are known to divide asymmetrically to produce daughter cells with
different fates. In these divisions, it is proposed that the position from
which Wnt signaling is presented, i.e. from posterior to the cells in both
cases, regulates cell fate polarization and spindle orientation (e.g.
Thorpe et al., 1997;
Goldstein et al., 2006). In the
Drosophila peripheral nervous system, sensory organ precursor cells
undergo several rounds of asymmetric division within the plane of the
epithelium. The cell fate polarization and spindle orientation depends on the
planar cell polarity within the epithelium
(Bellaiche et al., 2004),
although daughter cells further communicate with each other through Notch
signaling to assume mutually exclusive fates
(Rhyu et al., 1994). In
contrast, examples using intrinsic cues are found in neuroblasts in the
Drosophila central nervous system (e.g.
Betschinger and Knoblich,
2004). In vertebrate skin development, the basal epidermal cells
also use their intrinsic polarity and divide asymmetrically, generating a
committed suprabasal cell and a proliferative basal stem cell
(Lechler and Fuchs, 2005).
Thus, asymmetric cell division is a fundamental system for establishing
various cell types during development; however, the way in which mother cells
polarize varies.
In ascidian embryos, asymmetric cell divisions occur at the division to the
64-cell stage in the vegetal marginal blastomeres, which encircle the central
endoderm located at the vegetal pole (Fig.
1). At this division, four posterior blastomeres (pairs of the
B6.2 and B6.4; mesenchyme/muscle mother blastomeres) divide into four outer
muscle precursors and four inner mesenchyme precursors. Similarly, in the
anterior region, four blastomeres (pairs of the A6.2 and A6.4; notochord/nerve
cord mother blastomeres) divide to produce four outer nerve cord precursors
and four inner notochord precursors
(Nishida, 1987). These
asymmetric cell divisions are known to involve the FGF signal from the
endoderm, and the differing ways to respond to FGF signal between the anterior
and posterior regions are accounted by the presence and absence of maternally
localized factor, macho-1 (Fig.
1C) (reviewed by Nishida,
2005). When the inductive influence is inhibited by isolation of
the blastomeres or using inhibitors of FGF signaling, both daughters of the
mesenchyme/muscle and notochord/nerve cord precursors assume the default
muscle and nerve cord fates, respectively
(Kim et al., 2000;
Minokawa et al., 2001;
Kim and Nishida, 2001). In
contrast, when the mother precursors are isolated and treated with FGF protein
over their entire surface, both the daughter cells adopt the induced
mesenchyme and notochord fates (Nakatani
et al., 1996; Kim et al.,
2000).
The occurrence of the signaling prior to, but not after, cell division that
segregates two distinct fates into two daughters, is one of the most important
criteria as to whether the division can be regarded as induced asymmetric cell
division. Several lines of evidence indicate that induction of notochord and
mesenchyme fates by the FGF signal takes place during the 32-cell stage, right
before the division that separates induced and default fates into daughter
cells. In the nerve cord/notochord division, recombination of isolated nerve
cord/notochord and endoderm (inducer) precursor blastomeres resulted in
notochord formation only when the nerve cord/notochord precursors to be
recombined were in the initial two thirds of the cell cycle at the 32-cell
stage (Nakatani and Nishida,
1999). The nerve cord/notochord precursors lose their competence
at the end of that stage. It is also shown that the inducing ability of the
endoderm blastomeres persists even after the 64-cell stage. Consistently, the
nerve cord/notochord precursors lose their competence to respond to the
treatment of FGF protein at the division to the 64-cell stage
(Nakatani et al., 1996). In
addition, the sensitive period to the FGF receptor and MEK inhibitors ends at
the sixth cleavage, suggesting that notochord induction completes during the
32-cell stage (Kim and Nishida,
2001). As to the division of the muscle/mesenchyme precursors, the
precursors acquire the ability of autonomous mesenchyme formation in isolation
sometime in the last half of the cell cycle at the 32-cell stage, indicating
that mesenchyme induction also completes during that stage
(Kim and Nishida, 1999).
Accordingly, both precursor blastomeres appear to be polarized by the external
signal and divide to produce daughter cells with distinct fates through
asymmetric divisions.
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;
Goldstein, 1993;
Goldstein, 1995a;
Goldstein et al., 2006). The
EMS divides to produce the E cell close to its inducer P2 cell and
the MS cell away from it. Importantly, induction of the E cell by Wnt
signaling from the P2 cell takes place before the EMS divides. The
daughters of the EMS adopt default MS fate in the absence of Wnt signaling.
Recently, it was shown that the polarity of the EMS is determined by the
direction from which Wnt signaling is presented
(Goldstein et al., 2006).
Therefore, similarly to what has recently been demonstrated in C.
elegans, a model of directed-signaling-mediated asymmetric division has
been proposed in the ascidian, in which the polarity of the asymmetric
divisions is determined by the direction from which the FGF signal comes
(Nishida, 2002).
It is, however, still unknown whether the polarities are determined solely
by the FGF signal, and/or whether their internal state or other cell
interactions are also involved. In particular, the notochord fate can not only
be induced by endoderm blastomeres but also by neighboring notochord/nerve
cord blastomeres, while mesenchyme/muscle precursors do not have an ability to
induce the mesenchyme fate mutually
(Nakatani and Nishida, 1994;
Kim and Nishida, 1999). In
agreement with this, FGF9/16/20 is expressed in nerve cord/notochord
precursors as well as in endoderm cells, but not in precursors of
mesenchyme/muscle (Fig. 1C)
(Imai et al., 2002a;
Kumano et al., 2006). These
findings suggest that not only the side of the nerve cord/notochord precursor
cells on which notochord precursors arise (endoderm side) but also the other
sides apart from the endoderm could be exposed to the FGF signal, and yet only
one daughter cell is induced to become notochord. In addition, the brain is
known to be induced by FGF signal from the nerve cord/notochord precursors
(Fig. 1C,D)
(Bertrand et al., 2003). As
brain precursors are situated on the opposite side of the nerve cord/notochord
precursors from the endoderm, this clearly implies that the A6.2
notochord/nerve cord blastomere, for example, would receive the FGF signal
from every direction. Therefore, it is difficult to conceive how it could be
polarized solely by the FGF signal.
In this study, using molecular and micromanipulative approaches, we investigated whether the asymmetries are determined solely by the direction from which the FGF signal is presented. Our results show that whereas the polarity of the muscle/mesenchyme mother cells is indeed determined in this way, establishment of the polarity in the nerve cord/notochord mother cells depends on a possible antagonistic action between the FGF signal and a suppressive signal from the anterior ectoderm, providing a newly characterized mechanism underlying asymmetric cell divisions.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Blastomere manipulation and inhibition of cell division
Fertilized eggs were manually devitellinated with tungsten needles and
reared in 0.9% agar-coated plastic dishes filled with seawater. Identified
blastomeres were isolated from embryos with a fine glass needle under a
stereomicroscope (SZX-12; Olympus). Isolated blastomeres were cultured
separately until they developed into partial embryos. For recombination, an
isolated group of blastomeres was made to adhere to another group by mutual
adhesiveness. For blastomere removal and transplantation at the early 32-cell
stage, ectodermal blastomeres (descendants of a4.2 or b4.2) were removed from
a host embryo with a fine glass needle. Donor blastomeres that had been
isolated from another early 32-cell embryo were immediately transplanted to
the region of the host embryo from which the ectoderm cells had been removed.
In some experiments, cleavage was permanently arrested with 2.5 µg/ml
cytochalasin B (Sigma) from the 110-cell stage onward. Embryos were cultured
and fixed at appropriate stages for immunohistochemistry and in situ
hybridization.
Injection of MOs and synthetic mRNAs
An antisense morpholino oligonucleotide (MO; Gene Tools) complementary to
Hr-FGF9/16/20 (5'-TACCATTTGTACTGAAGGCATTTTC-3')
(Kumano et al., 2006) was used
to suppress its translation. In control experiments, we used standard control
MO supplied by the manufacturer. Hr-FGF9/16/20 and Hr-FoxA
plasmids for in vitro RNA synthesis were prepared by PCR-amplifying fragments
and subcloning them into the pBluescript-HTB(N) vector
(Kumano et al., 2006). As a
control for RNA injection, a venus YFP fragment was subcloned into
pBluescript-RN3 (Lemaire et al.,
1995). Capped Hr-FGF9/16/20, Hr-FoxA and venus
YFP mRNAs were synthesized with the mMessage mMachine kit (Ambion) and
subsequently Poly(A) was added with a Poly(A) Tailing kit (Ambion). MOs (30
pg), synthetic mRNAs (10 pg with the exception of Hr-FoxA mRNA: 0.5-1
pg) or both were injected into blastomeres at the eight-cell stage.
Microinjection was carried out as described previously
(Miya et al., 1997).
Immunohistochemistry and whole-mount in situ hybridization
Formation of mesenchyme was monitored at the equivalent of the larval stage
(about 10 hours after the hatching stage) by staining with the anti-Mch-3
monoclonal antibody in embryos whose cleavage was arrested at the 110-cell
stage. The antibody specifically recognizes small particles in mesenchyme
cells of Halocynthia larvae (Kim
and Nishida, 1999). The specimens were fixed for 10 minutes in
methanol at -20°C. Indirect immunofluorescence detection was carried out
by standard methods using a TSA fluorescein system (PerkinElmer Life Sciences)
according to the manufacturer's protocol. Immunostaining for activated MAPK
(antidiphosphorylated ERK1/2, M8159; Sigma) and nuclear staining with DAPI
were performed as described by Nishida
(Nishida, 2003).
Whole-mount in situ hybridization was performed according to Miya et al.
(Miya et al., 1997). Specimens
were hybridized by using digoxigeninlabeled Hr-MA4, Hr-Bra, Hr-FoxA
and Hr-ETR1 antisense probes. Hr-MA4, encoding the muscle
actin gene, was used to assess muscle specification
(Satou et al., 1995).
Hr-Bra, encoding the Brachyury gene, was used for notochord
specification (Yasuo and Satoh,
1993). Hr-FoxA (formerly Hr-HNF3-1) encodes a
homolog of class I fork head/HNF-3
(Shimauchi et al., 1997). The
expression of these genes was monitored at the 110-cell stage.
Hr-ETR1, encoding an RNA-binding protein of the Elav family, was used
as a molecular marker for nerve cord specification
(Minokawa et al., 2001), and
its expression was monitored at the neural plate stage in embryos where
cleavage was arrested at the 110-cell stage.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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We used the Mch-3 antigen as a marker of mesenchyme differentiation. When cleavages were permanently arrested at the 110-cell stage, the Mch-3 antigen was expressed in 3.2 out of the four (two on each side) mesenchyme-lineage blastomeres on average (Fig. 2C). In endoderm-transplanted embryos, expression of the Mch-3 antigen was detected not only in cells of mesenchyme lineage, but also ectopically in cells of muscle lineage on the left (transplanted) side (Fig. 2D, arrowhead). The number of Mch-3-positive cells was 4.4 on average. Transplantation of isolated b-line ectodermal (epidermal) cells as a control experiment had no effect on Mch-3 expression (3.1 on average, Fig. 2E). The expression of muscle actin was monitored at the 110-cell stage and found to be abrogated when endoderm was transplanted (Fig. 2G), whereas it was unaffected by transplantation of ectodermal cells (Fig. 2H). Therefore, transplantation of endoderm cells to the side opposite from the host endoderm causes muscle lineage cells to assume a mesenchyme fate.
In the case of notochord and nerve cord, a-line ectoderm cells were replaced by endoderm cells of a different embryo at the early 32-cell stage so that the notochord/nerve cord precursors became sandwiched by endoderm cells (Fig. 2B). The expression of Hr-Bra, a marker of notochord differentiation, monitored at the 110-cell stage was detected ectopically in cells of nerve cord lineage (Fig. 2J, arrowheads). Transplantation of a-line ectoderm as a control scarcely induced the expression of Hr-Bra (Fig. 2K). The expression of Hr-ETR1, a marker of neural plate, including nerve cord as well as brain, was abrogated in cells of nerve cord lineage in the cleavagearrested 110-cell embryos when endoderm was transplanted (Fig. 2M). The expression in brain precursors was also absent because brain-lineage cells were replaced with transplanted endoderm cells. Hr-ETR1 expression in ectoderm-transplanted control embryos was comparable to that in unoperated control embryos (Fig. 2N). Accordingly, as is the case for muscle/mesenchyme, transplantation of endoderm cells to the side opposite from the host endoderm induces a notochord fate in the nerve cord lineage.
Polarity of asymmetric division to segregate mesenchyme and muscle fates is determined solely by FGF signaling
Our final goal was to reverse the polarity of asymmetric cell divisions by
manipulating the position of the signal source, which would work if polarity
is indeed determined solely by the direction from which the signal molecule
comes. We exploited the recent isolation of the Halocynthia ortholog
of FGF9/16/20 (Kumano et al.,
2006). Knockdown of Hr-FGF9/16/20 by injecting antisense
MO into the left A4.1 and B4.1 blastomeres of the eight-cell embryo resulted
in almost complete loss of Mch-3 antigen expression in mesenchyme lineage
cells, with concomitant ectopic expression of muscle actin in these
cells (Fig. 3A,A').
Consistent with this, transplantation of endoderm cells from such morphants
did not alter the patterning (Fig.
3C,C'), whereas transplantation of endoderm cells from
embryos injected with control MO sustained the inducing ability (data not
shown). In addition, when b4.2 on the left side was injected with synthetic
Hr-FGF9/16/20 mRNA at the eight-cell stage, the cells of the muscle
lineage in the left half assumed a mesenchyme fate with concomitant loss of
muscle fate (Fig. 3B,B').
These results suggest that FGF9/16/20 is indeed an endogenous inducer for
mesenchyme induction.
In an attempt to promote asymmetric division with reversed polarity, we performed a double injection, in which A4.1 and B4.1 were injected with FGF MO and b4.2 with FGF mRNA, simultaneously. Such embryos, however, had Mch-3 expression in both muscle and mesenchyme lineages (data not shown), probably because the effect of the injected mRNA was too strong. To overcome this problem, we transplanted b-line ectoderm cells that had been injected with FGF mRNA at the eight-cell stage to the bline ectoderm-removed region of another embryo in which FGF9/16/20 had been knocked down (Fig. 3, diagram above panels D-G). Although transplantation of all the descendants of left-side bline blastomeres still had an excessive effect (Fig. 3D,D'), when part of the descendants was used, it resulted in perfect reversal of the fates (Fig. 3E,E'), as shown on the left side of Fig. 3E'', although the percentage was not so high (approximately 30%). Control embryos, in which control MO and venus YFP mRNA were used, never showed such reversal (Fig. 3G,G'). In another control, transplantation of uninjected b4.2 ectoderm cells did not induce expression of the mesenchyme marker in FGF-knockdown embryos, nor did it block muscle fate (Fig. 3F,F'). Finally, as a simple control, embryos with b-line ectoderm cells just removed at the early 32-cell stage differentiated mesenchyme and muscle cells in their normal positions (data not shown). Taken together, these results suggest that the polarity of asymmetric division of muscle/mesenchyme mother cells depends simply on the direction from which the FGF9/16/20 signal is presented. The FGF9/16/20 signal is interpreted equally on the future mesenchyme and muscle sides during the 32-cell stage, suggesting that no intrinsic cues are involved in the process.
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).
However, knockdown of Hr-FGF9/16/20 locally only in the descendants
of one A4.1 blastomere by injecting the left-side A4.1 with MO at the
eight-cell stage resulted in partial loss of Hr-Bra expression in the
descendant cells (Fig. 4A,
black arrowheads). The cells that still expressed Hr-Bra were, in
most cases, medial notochord precursors (yellow arrowheads) derived from a
single A6.2 at the 32-cell stage. It is likely that the medial cell was
induced at the 32-cell stage across the midline by the right-side A6.2
blastomere, as notochord blastomeres can induce each other
(Nakatani and Nishida, 1994).
This is not consistent with our hypothesis that the FGF signal alone
determines the polarity, because the left-side A6.2 cell received the FGF
signal only from the right side and not from the endodermal side but still
underwent asymmetric division with normal polarity. As for the nerve cord
marker, only two lateral cells of the notochord lineage that did not express
Hr-Bra showed ectopic Hr-ETR1 expression
(Fig. 4A',
arrowheads). Injection of the left-side a4.2 blastomere with FGF mRNA at the eight-cell stage resulted in only two lateral cells of nerve cord lineage expressing Hr-Bra weakly (Fig. 4B, red arrowheads), and resulted in great reduction in Hr-ETR1 expression in nerve cord lineage (Fig. 4B'). The expression of Hr-ETR1 was observed in a broad area in the animal hemisphere, probably because of broad induction of brain fate by overexpressed FGF. Double injection of MO and mRNA in an attempt to reverse the polarity, in which the left-side A4.1 and a4.2 blastomeres were injected with FGF MO and mRNA, respectively, at the eight-cell stage did not work again (Fig. 4C,C') and resulted in the same expression pattern as that observed after single FGF mRNA injection. Overexpression of FGF in the animal hemisphere restored Hr-Bra expression mostly in notochord precursors without promoting it in the nerve cord lineage in the FGF-less background (compare Fig. 4C with 4A). Embryos injected with control MO and venus YFP mRNA showed normal gene expression (Fig. 4D,D').
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Ectodermal signal antagonizes activation of MAPK and FoxA gene expression
In order to identify what might be suppressed by the signal from the
ectoderm, we examined MAPK (ERK) activation with an antibody against
diphosphorylated MAPK in a-line ectoderm-removed embryos. In normal control
44-cell embryos, activation of MAPK was observed in notochord precursors in
all cases (pink letters in Fig.
6A,A', n=42), whereas it was observed in nerve cord
precursors only in 26% of cases (white letters and arrowheads in
Fig. 6A,A'), as reported
previously (Nishida, 2003).
This is consistent with the fact that FGF signaling transduces the signal via
MAPK. The diphosphorylated MAPK signal observed in nerve cord, if any, was
weaker than that in notochord and was restricted to the two medial precursors
in most cases. In contrast, in embryos depleted of a-line ectoderm, MAPK was
activated in nerve cord precursors (yellow arrowheads) as well as in notochord
precursors in 89% of cases (pink letters in
Fig. 6B,B',
n=44, P<0.01). The signal in nerve cord precursors was
evident in the two medial cells and weaker in the two lateral cells. These
observations indicate that the ectodermal signal antagonizes FGF signaling by
suppressing activation of MAPK in nerve cord precursors.
Previous studies have shown that transcription factors, FoxA (formerly
HNF3-1) and ZicN (ZicL in Ciona savignyi), are essential for
induction of Brachyury by FGF as intrinsic competence factors in
signal-receiving cells (Shimauchi et al.,
1997; Wada and Saiga,
2002; Imai et al.,
2002b; Imai et al.,
2006; Yagi et al.,
2003; Kumano et al.,
2006). These factors are expressed in notochord/nerve cord
precursors at the 32-cell stage. At the 64- and 110-cell stages, however,
Hr-ZicN is continuously expressed in both the notochord and nerve
cord precursors and required for execution of both fates, while expression of
Hr-FoxA is downregulated in nerve cord precursors
(Kumano et al., 2006). To
clarify if the ectoderm signal affects Hr-FoxA expression in nerve
cord lineage cells, we examined the expression of Hr-FoxA in a-line
ectoderm-removed embryos. In contrast to control 110-cell embryos, in which
Hr-FoxA was expressed in precursors of notochord, endoderm and trunk
lateral cells (Fig. 6C),
embryos without a-line ectoderm cells showed Hr-FoxA expression in
cells of nerve cord lineage in addition to the precursors mentioned above
(Fig. 6D). These results
suggest that the ectodermal signal suppresses Hr-FoxA expression in
the nerve cord lineage. As overexpression of Hr-FoxA mRNA did not
cause ectopic Hr-Bra expression by itself in nerve cord lineage
(Kumano et al., 2006),
activation of Hr-Bra expression by MAPK and its downstream Ets is
also likely suppressed by the ectodermal signal.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Direction of FGF9/16/20 signal polarizes asymmetric division of mesenchyme/muscle blastomeres
In this study, we successfully reversed the polarity of muscle/mesenchyme
precursors by manipulating the position of FGF signal sources
(Fig. 7B). Thus, a directed
FGF9/16/20 signal from endoderm is the key regulator of the polarity of the
asymmetric division that segregates mesenchyme and muscle fates. Our previous
work has shown that all the neighboring blastomeres in contact with
mesenchyme/muscle precursors at the 32-cell stage, with the exception of
endoderm precursors, do not have the ability to induce a mesenchyme fate
(Kim and Nishida, 1999). In
accordance with this, only endoderm precursors express FGF9/16/20 at
that stage (Fig. 1C).
Therefore, mesenchyme/muscle precursors receive the FGF signal only from the
endodermal side. The asymmetric divisions of the EMS and T cells in C.
elegans are known to utilize similar mechanisms, whereby the position
from which Wnt signaling is presented determines the cell polarities
(Goldstein et al., 2006). As
ascidians and C. elegans have similar modes of asymmetric cell
division, both involving fate determination by inductive signals immediately
before division, directed-signaling-mediated asymmetric cell division could be
a basic mechanism for producing a variety of cell types during animal
development, especially when the embryos consist of a small number of
cells.
However, the asymmetric cell divisions in ascidians and C. elegans
differ on two points. First, both daughter cells in ascidian embryos assume
induced fate when the mother cells receive the inductive signal
bi-directionally from opposite sides. This happens when isolated blastomeres
are treated by FGF protein over the entire surface
(Nakatani et al., 1996) and
when mother cells are sandwiched by the host and transplanted endoderm cells
as well as by host endoderm and transplanted FGF-over-expressing cells
(present study). In C. elegans, in contrast, if EMS is signaled from
opposite sides by flanking it with two P2 blastomeres, only rarely
do both daughters adopt the induced E fate; rather, only one daughter adopts
an E fate (Goldstein, 1995a).
Goldstein has proposed a model whereby a signal from P2 may cause a
segregation of cytoplasmic components, making one daughter differ from the
other. Accordingly, induction on one side might interfere with induction on
the other side. The second difference is whether the directed signals regulate
spindle orientation during the asymmetric divisions. In C. elegans,
orientation of the mitotic spindle is coordinated with the polarity of cell
fate asymmetry and ensures that different fates are segregated into different
daughters (Goldstein, 1995b;
Goldstein, 2000). In ascidians,
however, blockade of FGF signaling by treatment with a MEK inhibitor or an FGF
receptor inhibitor, or by injection of FGF MO does not alter the
cleavage pattern up to the 64-cell stage
(Kim and Nishida, 2001;
Kumano et al., 2006) (present
study). Thus, it is likely that FGF signaling is not involved in orientation
of the mitotic spindles and cleavage planes in ascidian embryos.
Suppression of notochord fate in nerve cord precursors by an ectodermal signal
In contrast to the situation in muscle/mesenchyme mother cells, that in
notochord/nerve cord blastomeres was thought to be more complicated because
the mother cells are likely to be exposed to the FGF signal not only from the
endoderm side but also from every tangential direction
(Fig. 7C). The present results
also support the idea of the dispensability of the FGF signal from endoderm.
Therefore, the embryos must have evolved other ways of accomplishing
polarization of notochord/nerve cord blastomeres by additional signals. In the
present study, we demonstrated the presence of an additional signal that is
secreted from the anterior ectoderm and controls the polarization. This
influence from ectoderm completes its role by the early 64-cell stage. The
discovery of this signal clarifies the complex situation required for
asymmetric division and is still consistent with findings from previous
experiments. For example, when an isolated notochord/nerve cord blastomere
receives the signal by treatment with basic FGF protein, both daughter cells
adopt a notochord fate (Nakatani et al.,
1996; Minokawa et al.,
2001), probably because of exposure to the FGF signal over the
entire cell surface in the absence of the ectodermal signal. Furthermore, in a
simplified experimental situation, namely in the absence of the ectodermal
signal, notochord/nerve cord blastomeres seem to be polarized solely by the
direction from which the FGF signal comes, as observed in mesenchyme/muscle
blastomeres. When a single notochord/nerve cord blastomere was co-isolated or
recombined with a single endoderm blastomere, only half of the descendants of
the notochord/nerve cord blastomere expressed Hr-Bra
(Nakatani et al., 1996). In
this case, the notochord/nerve cord blastomere receives an FGF signal only
from the endodermal side but not from the other sides, and is likely to be
polarized solely by the FGF signal.
In normal embryos, the medial notochord/nerve cord blastomere (A6.2 in Fig. 1C) is likely to receive more FGF signal than the lateral one (A6.4) at the 32-cell stage when induction occurs because the medial cell is in contact with three FGF-expressing cells, namely another medial cell across the midline, the lateral cell and an endoderm cell, while the lateral cell is only adjacent to two, the medial cell and an endoderm cell. Consistent with this, weak activation of MAPK was occasionally observed in two medial nerve cord precursors at the 44-cell stage. This might be a reason why, in anterior ectoderm-removed embryos, ectopic diphosphorylated MAPK and Bra expression was often detected only in the descendants of the two medial nerve cord precursors and Hr-ETR1 remains expressed in the lateral nerve cord precursors in most cases (Fig. 4E, Fig. 5A,A', Fig. 6B). Similarly, there appeared to be a difference in the intensity of, or sensitivity to, the ectodermal signal between the medial and lateral sides for some unknown reason, because when we overexpressed FGF in animal blastomeres, only lateral nerve cord cells showed weak Hr-Bra expression (Fig. 4B, Fig. 6E). These observations suggest that the presence of ectodermal signal is more crucial for generation of the medial nerve cord precursors than that of the lateral precursors.
Ectodermal signal suppresses activation of MAPK and expression of FoxA
The signal from anterior ectoderm suppressed the activation of MAPK in
nerve cord lineage cells. The FGF signal activates the Ras-MEK-MAPK-Ets
signaling pathway in ascidian embryos (Kim
and Nishida, 2001; Miya and
Nishida, 2003; Bertrand et al.,
2003). Thus, it is plausible that the ectodermal signal
antagonizes a step(s) of FGF signal transduction.
Our results suggested that the ectodermal signal also downregulates
FoxA expression in nerve cord lineage. Hr-FoxA has recently been
identified as an intrinsic competence factor for notochord induction
(Kumano et al., 2006). When
FoxA or FGF mRNA was solely overexpressed, the nerve cord
precursors scarcely expressed Bra. In contrast, overexpression of
FoxA in the A-line cells and FGF in the a-line cells was able to fully induce
an ectopic notochord fate in nerve cord lineage cells
(Fig. 6F). Therefore, the
ectodermal signal blocks information from both intrinsic and extrinsic cues
indispensable for Bra expression to ensure the asymmetry of
Bra expression after asymmetric division.
It remains to be determined whether this suppression of Hr-FoxA is
achieved through inactivation of MAPK, or of FGF signaling that does not
involve MAPK activation, or even an unknown pathway that is independent of FGF
signaling. It is noteworthy that expression of FoxA starts
cell-autonomously at the 32-cell stage, whereas its maintenance at the 64-cell
stage depends on FGF signaling (Kumano et
al., 2006). Also, it has been proposed that the regulation of
Hr-Bra expression by FGF signaling involves two distinct pathways:
FoxA-dependent and - independent pathways
(Kumano et al., 2006). As
Bra expression was fully induced in the nerve cord lineage only when
FoxA was co-expressed in the background in which FGF was overexpressed, the
threshold over which FGF signaling is able to activate Bra expression
in the FoxA-independent pathway might be lower than that for the activation of
FoxA expression. Likewise, expression of ETR-1 is more
sensitive to FGF signaling than that of FoxA, because ETR-1
expression was eliminated when FGF was overexpressed in a-line ectoderm
without FoxA expression fully activated in the nerve cord
lineage.
In conclusion, the polarities of the two kinds of asymmetric cell division in ascidian embryos are determined by extracellular cues from neighboring cells. In mesenchyme/muscle precursors, the polarity is simply specified by the direction from which the FGF signal is presented. In notochord/nerve cord precursors, the mechanism is more complicated, as the polarity depends on a yetunknown signal from ectoderm that antagonizes the FGF signal. The presence of this additional signal in the asymmetric division of nerve cord/notochord mother cells reflects the fact that the notochord/nerve cord precursors need to secrete FGF in order to induce brain in the adjacent ectoderm cells.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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