? Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuta,
Yokohama 226-8501, Japan
* Present address: Department of Zoology, Graduate School of Science, Kyoto
University, Sakyo-ku, Kyoto 606-8502, Japan
Author for correspondence (e-mail:
hnishida{at}bio.titech.ac.jp)
Accepted 2 April 2003
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SUMMARY |
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Suppression of the function of macho-1, a muscle determinant in ascidian eggs, by antisense oligonucleotide was enough to allow autonomous endoderm specification. Therefore, the cell interactions induce endoderm formation by suppressing the function of macho-1, which is to promote muscle fate. These findings suggest the presence of novel mechanisms that suppress functions of inappropriately distributed maternal determinants via cell interactions after embryogenesis starts. Such cell interactions would restrict the regions where maternal determinants work, and play a key role in marking precise boundaries between precursor cells of different tissue types.
Key words: Ascidian embryos, Endoderm formation, Muscle determinants, Inductive interactions, FGF, BMP
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INTRODUCTION |
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Endoderm cells are present in the central part of the trunk region of
tadpole larvae (Fig. 1A). These
cells are homogeneous in appearance and rich in yolk granules, and probably
provide the embryos with nutrients. Endoderm cells in Halocynthia do
not undergo terminal differentiation during larval development, because
Halocynthia tadpoles do not feed. After metamorphosis, larval
endoderm cells mainly give rise to peribranchial epithelium, gill and
digestive organs (Hirano and Nishida,
2000). However, during embryogenesis, endoderm cells start some
differentiation processes and initiate to express endoderm-specific alkaline
phosphatase (ALP) (Minganti,
1954
; Whittaker,
1977
; Nishida and Kumano,
1997
; Kumano and Nishida,
1998
). All of the endoderm cells of a larva are derived from the
vegetal blastomeres of an eight-cell embryo namely the anterior A4.1
cell pair and the posterior B4.1 cell pair
(Fig. 1A). By contrast,
blastomeres of the animal hemisphere do not produce endoderm.
|
Glycogen synthetase kinase 3 (GSK3) and the ß-catenin signaling
pathway play crucial roles in maternal mechanisms that specify the
animal-vegetal axis in sea urchin
(Wikramanayake et al., 1998;
Emily-Fenouil et al., 1998
;
Logan et al., 1999
). Imai et
al. (Imai et al., 2000
) have
reported a role for ß-catenin in the specification of vegetal fate in
embryos of the ascidians Ciona intestinalis and C. savignyi.
In this regard, ascidian embryos show similarity to echinoderm embryos. The
authors reported preferential ß-catenin nuclear localization in the
vegetal hemisphere in cleavage stage embryos. When mRNA encoding the
stabilized form of ß-catenin was injected into eggs, nuclear
ß-catenin was observed also in the animal hemisphere. In these embryos,
most embryonic cells expressed ALP. To inhibit ß-catenin function in
nuclei, ß-catenin was sequestered to a cell adhesion complex by
overexpression of cadherin. Nuclear staining with ß-catenin antibody was
abolished in the entire embryo and ALP expression was lost. These observations
indicate that animal-vegetal axis specification is mediated by ß-catenin
signaling. ß-catenin is not localized in eggs and early cleavage stage
embryos; so localized endoderm determinants would be molecules that stabilize
ß-catenin in the vegetal hemisphere.
Early zygotic events during endoderm formation have been well analyzed in
nematodes, Xenopus, zebrafish and mammals
(Hudson et al., 1997;
Zaret, 1999
;
Aoki et al., 2002
;
Maduro and Rothman, 2002
). In
ascidian embryos, the expression of a LIM class homeobox gene, Hrlim,
starts in endoderm precursors at the 32-cell stage, although it is not
restricted to endoderm (Wada et al.,
1995
). In Ciona savignyi, the ortholog, Cs-lhx3,
lies downstream of ß-catenin and is responsible for ALP expression in
endoderm (Satou et al., 2001
).
At the 76-cell stage, expression of the TITF1 homologs of Ciona
intestinalis and C. savignyi, Cititf1 and Cs-ttf1, is
initiated exclusively in endoderm precursors. TITF1 is a transcription factor
containing an NK-2-like homeodomain
(Lazzaro et al., 1991
;
Kimura et al., 1996
). When
synthetic mRNA of Cititf1 and Cs-ttf1 is injected into eggs,
ectopic expression of ALP is promoted in non-endoderm cells
(Ristoratore et al., 1999
;
Satou et al., 2001
).
macho-1 mRNA has been identified as a localized maternal muscle
determinant within ascidian egg cytoplasm
(Nishida and Sawada, 2001).
macho-1 encodes a putative transcription factor that has zinc-finger
domain. The presence of macho-1 protein promotes muscle fate. However, macho-1
products are inferred to be also present in mesenchyme precursor cells, and
macho-1-directed muscle fate must be suppressed by FGF signaling for proper
formation of mesenchyme cells (Kim and
Nishida, 1999
; Kim et al.,
2000
). In this study, we demonstrate that muscle fate directed by
macho-1 should also be suppressed by cell interactions in the posterior
endoderm. These observations suggest the presence of novel mechanisms that
suppress functions of inappropriately distributed maternal determinants via
cell interactions after embryogenesis starts.
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MATERIALS AND METHODS |
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Isolation and dissociation of blastomeres
Eggs were manually devitellinated with sharpened tungsten needles, and
reared in 0.9% agar-coated plastic dishes filled with sea water. Blastomeres
were identified and isolated from embryos by use of a fine glass needle under
a stereomicroscope. Isolated blastomeres were cultured separately in
agar-coated plastic dishes. Partial embryos were cultured until control
embryos reached hatching stage, and they were then prepared for histochemical
and immunohistochemical staining to detect endoderm and muscle formation. For
dissociation of embryonic cells, isolated blastomeres were incubated in
Ca2+-free artificial sea water (CFSW), such that daughter cells
were continuously separated. CFSW consisted of 460 mM NaCl, 9.3 mM KCl, 48 mM
MgSO4·7H2O, 6 mM NaHCO3, and 0.2 mM
ethylene-bis(oxyethylenenitrilo)-tetraacetic acid (EGTA). Dissociation was
monitored at frequent intervals and facilitated by gentle pipetting.
Dissociated cells continued to divide at normal rates. After the period of
dissociation, the cells were transferred separately to normal sea water and
allowed to develop into multicellular partial embryos. In some experiments,
cleavage of embryos was permanently arrested by treatment with 2 µg/ml
cytochalasin B (Sigma) at the 110-cell stage.
Treatment with growth factors and MEK inhibitor
Isolated or dissociated blastomeres were transferred into sea water that
contained 0.1% bovine serum albumin (BSA; Sigma) and 4 ng/ml bFGF protein
(Amersham) or 50 ng/ml BMP4 protein (R&D Systems). The concentrations of
FGF and BMP are effective enough to induce notochord formation in
Halocynthia (Nakatani et al.,
1996; Darras and Nishida,
2001
). In controls, blastomeres were treated with BSA in sea
water. To inhibit activation of the FGF-MAPK cascade, embryos were treated
with 1.3-2.0 µM SU5402 (Calbiochem) or 2 µM U0126 (Promega) from the
eight-cell stage to fixation. SU5402 belongs to a family of FGF signaling
inhibitors that bind specifically to the active sites of FGFR kinase domains
(Mohammadi et al., 1997
).
U0126 is an MEK inhibitor that inhibits phosphorylation and thereby activation
of MAPK by MEK (Favata et al.,
1998
). Both inhibitors work well in Halocynthia embryos
(Kim and Nishida, 2001
). In
controls, blastomeres were treated with 0.02% DMSO, the solvent of SU5402 and
U0126.
Histochemistry, immunohistochemistry and in situ hybridization
To detect endoderm formation, histochemical staining for alkaline
phosphatase (ALP) activity was carried out as described by Meedel and
Whittaker (Meedel and Whittaker,
1989) with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as
substrate. The reaction deposits purple products. Muscle cells were
histochemically stained for acetylcholinesterase (AChE) by the method
described by Whittaker (Whittaker,
1980
) with acetylthiocholine iodide as substrate. The reaction
deposits brown products. The monoclonal antibody Mu-2 binds to the heavy chain
of ascidian myosin, and is specific for muscle cells in larvae
(Nishikata et al., 1987
;
Makabe and Satoh, 1989
).
Specimens were stained for indirect immunofluorescence with Mu-2 antibody by
using Alexa 488-conjugated secondary antibody (Molecular Probes) or a TSA
fluorescein system (PerkinElmer Life Sciences) according to the manufacturer's
protocol. Specimens were then mounted in 80% glycerol and examined under an
epifluorescence microscope. RNA probes for in situ hybridization of
HrMA4 were prepared with a DIG RNA labeling kit (Boehringer-Mannheim,
Germany). HrMA4 encodes larval muscle actin of Halocynthia
(Satou et al., 1995
).
Morpholino antisense oligonucleotide
The efficiency of morpholino antisense oligonucleotide (MO; Gene Tools),
which is complementary to macho-1, has already been tested in
Halocynthia (K.K. and H.N., unpublished). The MO was
5'-AATTGCAAAACACAAAAATCACACG-3', which covers the 5'-UTR of
macho-1 mRNA (GenBank Accession Number, AB045124). MO was dissolved in water,
and 100-300 pg was injected into each fertilized egg. In control
experiments, 300 pg of four-mismatch control oligonucleotide
(5'-AATTCCAAATCACAATAATCTCACG-3') was injected.
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RESULTS |
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In the first series of experiments, isolated blastomeres were collected and
dissociated together (Table 1).
In dissociation of B4.1 descendants, endoderm formation was significantly
reduced. The fate of each descendant of the B4.1 blastomere is shown in
Fig. 1B. After three cleavages,
two out of eight blastomeres assume endoderm fate in normal embryogenesis
(Nishida, 1987). The B7.6
blastomere gives rise to endodermal strand cells of the tip of the tail. But
in Halocynthia, these cells do not express ALP activity. Therefore,
in dissociation experiments, we expected that 25% (two out of eight) of
partial embryos would develop into ALP-expressing endoderm cells. However, we
observed ALP expression in only 6% of the partial embryos
(Table 1;
Fig. 2A). In dissociation of
A4.1 descendants, endoderm formed in 31% of the partial embryos. This result
is comparable with the expectation from the cell lineage of A4.1 (38%, three
out of eight).
|
|
Suppression of muscle fate is required for posterior endoderm
formation
Next, we addressed what kind of tissue type cells the presumptive endoderm
blastomeres developed into under dissociation. The major fates of the B4.1
blastomeres of the eight-cell embryos are endoderm, muscle and mesenchyme
(Fig. 1B). For mesenchyme
formation, inductive cell interaction is required, and presumptive mesenchyme
blastomeres assume muscle fate without induction in blastomere-isolation
experiments (Kim and Nishida,
1999). Another indicative results is that, in cell dissociation of
whole embryos, the proportion of partial embryos composed of muscle increases
to one fourth of total partial embryos
(Nishida, 1992
). Therefore, it
is probable that every dissociated B-line (posterior-vegetal) blastomere
assume muscle fate, and we examined the possibility.
In massive dissociation of B4.1 blastomeres, 92% of partial embryos showed
acetylcholinesterase activity, a muscle-specific enzyme
(Whittaker, 1980) and 91%
expressed muscle myosin, which was detected by Mu-2 monoclonal antibody
(Nishikata et al., 1987
)
(Table 1;
Fig. 2B). The proportion was
much higher than expected from the cell lineage (38%; three out of eight),
even if mesenchyme cells develop into muscle (totally 63%; five out of eight)
(Fig. 1B). In those partial
embryos, it seemed that every constituent cell of every partial embryo
developed into muscle cells (Fig.
2B). By contrast, in dissociation of A4.1 descendants, muscle
formation was never observed. In individual dissociation of B4.1 blastomeres,
all seven partial embryos expressed myosin in 14 sets of experiments, six
expressed myosin in three sets and four expressed myosin in one set. These
results indicate that most partial embryos derived from B4.1 blastomeres
assumed muscle fate, even though the B4.1 blastomere also has endoderm and
mesenchyme fates. We further confirmed that an absence of Ca2+ in
sea water during cell dissociation does not account for our results. We
manually dissociated cleaved blastomeres in normal sea water with a fine glass
needle instead of Ca2+-free sea water, and we obtained essentially
same results namely, loss of endoderm and excessive muscle formation
(data not shown).
Cell interactions take place at the 16- to 32-cell stages
We simply isolated endoderm lineage cells at various stages, B4.1 at the
eight-cell stage, B5.1 at the 16-cell stage and B6.1 at the 32-cell stage
(Fig. 1B). In most cases (100%
of 68 cases, 85% of 66 cases and 95% of 42 cases, respectively), the partial
embryos expressed ALP (see Fig.
4A for B4.1 partial embryo).
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|
We examined this possibility by treating dissociated blastomeres with basic
FGF (Table 3;
Fig. 3A,B). B4.1 isolates were
dissociated twice at the 16- and 32-cell stages in sea water containing 0.1%
BSA and 4 ng/ml bFGF protein, then washed thoroughly with sea water and
cultured as partial embryos. The concentration of bFGF is effective enough to
induce notochord and mesenchyme formation in Halocynthia
(Nakatani et al., 1996;
Kim et al., 2000
). In control
partial embryos treated only with BSA, 99% of them expressed muscle myosin and
only 2% expressed ALP. When partial embryos were treated with FGF, 22% of them
developed ALP activity. The proportion was fairly close to expectation from
the lineage (25%, one out of four). Muscle formation was greatly reduced to
21% (expectation is 75%, three out of four). Most ALP-negative partial embryos
consisted of very small cells that resembled mesenchyme cells
(Fig. 3A). Thus, FGF was
efficient at suppressing muscle fate and inducing endoderm fate in the B-line
blastomeres.
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|
To verify that FGF-MEK signaling acts early on the specification of the
posterior endoderm, fate conversion to muscle was examined by monitoring the
expression of muscle actin (HrMA4) gene at the 64-cell stage. In
Halocynthia embryos, muscle actin expression starts as early as
32-cell stage (Satou et al.,
1995). At the 64-cell stage, two muscle precursor blastomere pairs
(B7.4 and B7.8 pair) express actin. B4.1 partial embryos were fixed at the
64-cell stage, and actin expression was examined by in situ hybridization
(Fig. 4C,F,J). In control
partial embryos treated with DMSO, majority expressed actin in two blastomeres
per partial embryo as expected (no blastomeres in 3% of cases, one in 3%, two
in 64%, three in 23%, four in 7%; n=66). In 30% of cases, the number
of positive blastomeres exceeded two, probably owing to inappropriate
arrangement of blastomeres in the partial embryos, which would result in
failure of correct cell interactions. By contrast, partial embryos treated
with MEK inhibitor expressed actin gene in many blastomeres (two blastomeres
in 4% of cases, three in 1%, four in 30%, five in 24%, six in 41%;
n=91). Effect of the FGFR inhibitor was a bit weaker, but similar
results was obtained (no blastomeres in 1% of cases, one in 1%, two in 16%,
three in 15%, four in 31%, five in 22%, six in 13%; n=91). In
treatment with either inhibitor, the maximum number of positive blastomeres
per partial embryo was six, although the partial embryos should consists of
eight blastomeres after three cell divisions. However, even in normal embryos,
the most posterior muscle precursor blastomeres (B7.5 pair) do not initiate
actin expression by the 64-cell stage. This is probably due to the general
repression of zygotic gene expression in the two posterior blastomere pairs
that are the putative germline lineage cells in ascidian embryos
(Tomioka et al., 2002
). Taking
account of this, and even if the inhibitors caused two mesenchyme blastomeres
to assumed muscle fate, the observed number of positive blastomeres indicates
that some endoderm precursors initiated the expression of actin gene at the
64-cell stage in significant number of the partial embryos.
Suppression of macho-1 function is enough to allow posterior endoderm
formation
Cell interaction mediated by FGF-MAPK signaling is suggested to play a role
in suppression of muscle fate in posterior B-line endoderm precursors.
Maternal macho-1 mRNA has been identified as localized muscle
determinants in ascidian eggs (Nishida and
Sawada, 2001). The macho-1 product is necessary and sufficient for
the formation of primary muscle cells of tadpole larvae. The next question we
asked was whether suppression of the macho-1 function and consequent
suppression of muscle fate is enough for specification of endoderm fate in the
B-line. To do this, we injected morpholino antisense oligonucleotide (MO)
complementary to macho-1 mRNA into fertilized eggs. The efficiency of
the macho-1 MO has already been confirmed (K.K. and H.N.,
unpublished).
In control experiments, 300 pg of four-mismatch control oligonucleotide was injected into fertilized eggs, and B4.1 blastomeres were isolated at the eight-cell stage. Immediately after isolation, B4.1 blastomeres were treated with control DMSO or 2 µM MEK inhibitor until the larval stage (Table 4). Treatment with DMSO did not affect development of the B4.1 blastomeres, and both ALP and myosin were expressed in most of the partial embryos (Fig. 5A,B). Treatment with MEK inhibitor resulted in loss of endoderm, and every constituent cell of all partial embryos expressed myosin (Fig. 5E,F), confirming the previous results. Then 100-300 pg of MO was injected into eggs (Table 4). In DMSO-treated B4.1 partial embryos, ALP was expressed as in controls, but expression of myosin was completely inhibited, supporting the validity of macho-1 MO (Fig. 5C,D). In partial embryos treated with MEK inhibitor, ALP expression was observed in 63% of cases and there was no myosin expression (Fig. 5G,H). Although the proportion of ALP-positive embryos was a little lower than in controls, suppression of macho-1 function restored endoderm formation in the presence of MEK inhibitor. Therefore, if there is no macho-1 activity, FGF-MEK signaling is not required to suppress muscle fate, and endoderm fate is autonomously executed in the B-line lineage.
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BMP is a candidate for the redundant signal
A plausible candidate for the signaling molecule is BMP. In
Halocynthia, the HrBMPb gene (the ascidian BMP2/4
homolog) is expressed at and after the 44-cell stage in anterovegetal
blastomeres (anterior endoderm precursors: A7.1, A7.2 and A7.5 blastomeres;
trunk lateral cell precursor: A7.6 blastomeres;
Fig. 1A)
(Darras and Nishida, 2001). In
addition, BMP is a potent signaling molecule in notochord induction as well as
FGF (Darras and Nishida, 2001
).
Therefore, we examined whether BMP is also able to induce posterior endoderm
formation.
Similar to the above experiments used to test FGF, B4.1 isolates were dissociated twice at the 16- and 32-cell stages in sea water containing 0.1% BSA and 50 ng/ml BMP4 protein, then washed thoroughly with sea water and cultured as partial embryos (Table 3; Fig. 2C,D). The concentration of BMP4 is effective at inducing notochord in Halocynthia. When partial embryos were treated with BMP, 16% of them developed ALP activity. The proportion was close to expectation from the lineage (25%, one out of four). There is no statistical difference in ALP expression between FGF and BMP treatments (0.2<P<0.3, Table 3). Thus, BMP was also efficient at inducing endoderm fate in the B-line blastomeres. However, in contrast to FGF treatment, muscle formation was not significantly reduced, being observed in 84% (expectation is 75%, three out of four).
In the second set of experiments, B4.1 partial embryos were treated with BMP protein without cell dissociation. First, isolated B4.1 blastomeres were treated with BSA and DMSO. ALP expression was observed in all cases (n=5) (Fig. 6G). Second, B4.1 isolates was treated with BSA and MEK inhibitor (from eight-cell to larval stage). ALP expression was greatly reduced to 6% of cases (n=34), as observed in the previous experiments (Fig. 6H). Third, B4.1 isolates was treated with both BMP (from eight- to 64-cell stage) and MEK inhibitor. BMP restored ALP expression in 56% of cases (n=66) in the presence of MEK inhibitor (Fig. 6I). These results indicate that BMP is a possible candidate of the redundant signals, and MEK is not required for the signal transduction to induce endoderm by BMP.
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DISCUSSION |
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Maternal endoderm determinants and cell interactions
In our previous study, the expression of ALP was observed in continuously
dissociated whole embryos (Nishida,
1992). The observation led us to conclude that endoderm formation
is a cell-autonomous process. However, at that time we did not discriminate
the anterior and posterior endoderm. In this study, we isolated B4.1 and A4.1
blastomeres at the eight-cell stage and dissociated their descendants to
evaluate the role of cell interactions separately in each lineage. This
uncovered a remarkable difference in endoderm specification between the two
lineages. Only in the B-line are cell interactions required. The difference is
caused by the presence of macho-1 products in the posterior region of the
embryos, as discussed in the next section.
Experiments involving transfer of cytoplasm have been carried out to
demonstrate the presence and localization of endoderm determinants, by fusing
isolated blastomeres with cytoplasmic fragments
(Nishida, 1993;
Yamada and Nishida, 1996
). The
results suggested that endoderm determinants reside in the unfertilized egg
and are partitioned into both A-line and B-line endoderm-lineage blastomeres
during cleavages. When cytoplasm of B4.1 blastomeres was transferred into
presumptive epidermis blastomeres, it promoted ectopic endoderm formation. The
transferred cytoplasm probably also promoted ectopic cell interactions within
the partial embryos to which it was introduced.
The requirement for cell interactions does not exclude the possibility that
endoderm fate is basically specified by maternal localized endoderm
determinants. In the absence of macho-1 function, embryos could form the
posterior endoderm even when they were treated with MEK inhibitor. Imai et al.
(Imai et al., 2000) report a
role for ß-catenin in vegetal fate specification in Ciona.
Preferential ß-catenin nuclear localization is observed in cells of the
vegetal hemisphere, including B-line endoderm precursors. These observations
support the view that endoderm specification is essentially mediated by a
maternal mechanism even in the B-line, and that cell interactions overlie it
as a parallel process to suppress inappropriate muscle fate.
Suppression of inappropriately distributed macho-1 activity is
required for proper specification of B-line cell fates
Maternal macho-1 mRNA has been identified as localized maternal
muscle determinant within ascidian egg cytoplasm
(Nishida and Sawada, 2001).
macho-1 encodes a putative transcription factor that has a
zinc-finger domain. In this study, we demonstrated that muscle fate directed
by macho-1 should be suppressed by cell interactions in the posterior
endoderm. In the absence of macho-1 activity, endoderm specification was
autonomously executed. Without suppression of macho-1 function, all
descendants of the B4.1 blastomeres developed into muscle, and ALP expression
was totally suppressed. Probably, macho-1 intensively directs muscle fate and
overcomes the endoderm fate that is directed by ß-catenin signaling.
Cells may have mechanisms that prohibit an intermediate state between
different cell types and force one or another cell fate. This will be an
interesting issue for future study.
In mesenchyme precursor blastomeres, macho-1 products are inferred to be
also present, and macho-1-directed muscle fate must be suppressed by FGF
signaling for proper formation of mesenchyme cells
(Kim and Nishida, 1999;
Kim et al., 2000
). The
following observations further highlight the importance of suppression of
muscle fate in the posterior vegetal region. In the posterior region of the
vegetal hemisphere, precursor cells of trunk ventral cells (TVCs) are present
(Fig. 7). When the precursors
are isolated from embryos, they also differentiate into larval muscle cells
(Nishida, 1992
). Therefore,
suppression of muscle fate by cell interactions is also required for the
formation of the trunk ventral cells. This coincides well with our observation
that every descendant cell of the B4.1 blastomeres assumed muscle fate when
they were dissociated or treated with MEK inhibitor. Therefore, suppression of
muscle fate would be necessary in all of the non-muscle lineages within the
region derived from the B4.1 blastomere
(Fig. 7, pink area).
|
Our recent results with macho-1 MO indicate that macho-1 is also required for mesenchyme formation (K.K., K. Sawada and H.N., unpublished). Therefore, macho-1 directs muscle fate in muscle cells; its function is probably modified by FGF signaling to promote mesenchyme fate in mesenchyme cells; and in endoderm cells the function is totally suppressed (Fig. 7). FGF is involved in cell signaling in both mesenchyme and endoderm formation. There will probably be intrinsic differences in responsiveness to FGF between mesenchyme and endoderm blastomeres, and the localization of endoderm determinants could account for the differences.
Redundant signaling is involved in posterior endoderm induction
When the B4.1 blastomeres were isolated and then dissociated or treated
with inhibitors of FGF signaling, presumptive endoderm blastomeres assumed
muscle fate. Consistently, FGF was potent to induce posterior endoderm in cell
dissociation experiment. Recently, we have investigated the spatiotemporal
pattern of activation of MAPK during embryogenesis of Halocynthia,
using an antibody specific to the activated form of MAPK
(Nishida, 2003). Inconsistent
with the present results, phosphorylated and activated MAPK becomes detectable
in the nuclei of every endoderm blastomere including both of the B- and A-line
at the 44-cell stage when endoderm induction was completed. It has been also
shown that the activation of MAPK in endoderm blastomeres is suppressed by MEK
inhibitor treatment. In our previous study
(Kim and Nishida, 2001
), we
reported that treatment with the FGFR inhibitor (SU5402) did not suppress the
endoderm formation in isolated B-line blastomeres. In the experiments, embryos
was treated form the 2- to early 32-cell stage. The results were confirmed
again. It turns out that treatment up to the early 32-cell stage is not enough
to suppress endoderm formation because effects of FGF inhibitor is reversible
as shown by Kim and Nishida (Kim and
Nishida, 2001
), and that treatment should be continued at least up
to the 64-cell stage (data not shown). This is reasonable because the endoderm
induction take place during the 16- to 32-cell stage as revealed in the
present study.
However, the cleavage-arrest experiment indicated the presence of redundant
mechanisms in the induction of posterior endoderm. Both of FGF and BMP are
candidates for signaling molecules because both proteins were potent to induce
posterior endoderm in cell dissociation experiment. The FGF and BMP genes are
expressed at the right time and in the right place for posterior endoderm
induction during cleavage stages in ascidian
(Darras and Nishida, 2001;
Imai et al., 2002
). FGF is
expressed in both the anterior and posterior endoderm blastomeres, and BMP is
expressed in the anterior endoderm blastomeres. FGF signaling required MEK
activity, but BMP signaling did not, in accordance with the finding that BMP
signaling is transduced mainly by Smad proteins in various animals
(Whittman, 1998
; Massague and
Wotton, 2000). We tried to inhibit BMP signaling in cleavage-arrested and
MEK-inhibitor-treated embryos by injection of MO complementary to
HrBMPb. But it did not affect the endoderm and muscle formation. In
our experience, some MOs worked well but others did not, depending on genes
and targeted sequences in 5' UTR. Although BMP is a promising candidate
of signaling molecule secreted by anterior blastomeres, it is important to
further elucidate the role of endogenous HrBMPb gene by
loss-of-function type experiments in future study.
It is not clear how these distinct signaling mechanisms similarly promote endoderm formation. Treatment of dissociated blastomeres with FGF and BMP showed a slight difference. FGF treatment greatly reduced muscle formation, and most ALP-negative partial embryos seemed to develop into mesenchyme. In BMP treatment, however, ALP-negative partial embryos developed into muscle. This coincides with the observation that BMP is not effective in mesenchyme induction (H.N., unpublished). Therefore, FGF suppressed muscle fate in most blastomeres, but BMP is likely to suppress muscle fate only in presumptive endoderm blastomeres. Probably, the mechanism of suppression of muscle fate is different between FGF and BMP signaling.
There are similarities and differences between endoderm induction and
notochord induction in ascidians. FGF and BMP are both potent at inducing
notochord formation (Nakatani et al.,
1996; Darras and Nishida,
2001
). However, MEK is required for BMP to act in notochord
induction. Therefore, events during both kinds of induction would be similar
but not identical, although the details are unknown.
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ACKNOWLEDGMENTS |
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