Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
* Author for correspondence (e-mail: yutaka{at}ascidian.zool.kyoto-u.ac.jp)
Accepted 6 June 2003
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SUMMARY |
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We also demonstrate that the upstream regulatory mechanisms of Twist-like1 are different between B-line mesenchymal cells and the A-line mesenchymal cells called `trunk lateral cells'. FGF9/16/20 is required for the expression of Twist-like1 in B-line mesenchymal precursor cells, whereas FGF, FoxD and another novel bHLH factor called NoTrlc are required for Twist-like1 to be expressed in the A-line mesenchymal precursor cells. Therefore, two different but partially overlapping mechanisms are required for the expression of Twist-like1 in the mesenchymal precursors, which triggers the differentiation of the mesenchyme in Ciona embryos.
Key words: Ascidian, Ciona savignyi, Ciona intestinalis, Mesenchyme, Basic helix-loop-helix Transcription factors
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INTRODUCTION |
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The ascidian larval mesenchyme cells consist of about 900 cells that form
the adult body after metamorphosis (reviewed by
Satoh, 1994). The mesenchymal
cells are derived from the A7.6, B7.7 and B8.5 blastomere pairs of the
110-cell embryo (see Fig. 2A).
The mesenchymal cells derived from A7.6 blastomeres are called trunk lateral
cells (TLCs), and they mainly give rise to adult blood cells and body-wall
muscle (Nishide et al., 1989
;
Hirano and Nishida, 1997
). The
other two lineages of mesenchymal cells (B7.7 and B8.5) are mesenchymal cells
only in a narrow sense, and contribute mainly to tunic cells after
metamorphosis (Hirano and Nishida,
1997
). Although TLCs have been described as distinct from the
other two lineages of mesenchyme cells (B-line mesenchyme), numerous genes are
expressed specifically in both TLCs and B-line mesenchyme cells, suggesting
that these cells share some features
(Satou et al., 2001
;
Kusakabe et al., 2002
). In the
present study, we use the term `mesenchyme' for all of these cells, except
where indicated otherwise.
|
Ascidian larval development has been regarded as a `mosaic' development, in
which each tissue is specified by maternally supplied `determinants'.
Actually, the endoderm of Ciona is autonomously specified by
maternally supplied ß-catenin (Imai et
al., 2000). In another ascidian, Halocynthia roretzi,
muscle cells are autonomously specified by a maternally supplied Zic-related
transcription factor, macho1 (Nishida and
Sawada, 2001
). However, the specification of the remaining
mesodermal tissues, mesenchyme and notochord, requires inductive cell-to-cell
interaction. The notochord and the mesenchyme (except for TLCs) have been
demonstrated to be induced by presumptive endodermal cells around the 32-cell
stage (Nakatani and Nishida,
1994
; Kim et al.,
2000
). TLCs are induced by cells in the animal hemisphere at the
16-cell stage in Halocynthia
(Kawaminani and Nishida,
1997
). In a previous study, we demonstrated that a fibroblast
growth factor, Cs-FGF9/16/20 [previously called Cs-FGF4/6/9
(Imai et al., 2002a
); renamed
by Satou et al. (Satou et al.,
2002a
)], which is expressed in precursors for endoderm, TLCs,
notochord and nerve cord at the 16- and 32-cell stages, and in the nerve cord
and the muscle precursors at the 64- and 110-cell stages, is essential for the
induction of mesenchyme, including TLCs, in Ciona embryos
(Imai et al., 2002a
). However,
it is not yet known either when this FGF induces TLCs or from where the FGF
signal comes. In addition, the mechanism of the determination of mesenchymal
cells after induction has not been clarified yet. In the present study, we
identified genes for bHLH factors that work downstream of FGF9/16/20 and
determine the mesenchymal cell fate.
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MATERIALS AND METHODS |
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Isolation of cDNAs and sequence determination
Ciona intestinalis cDNA clones were obtained from the Ciona
intestinalis gene collection (Satou
et al., 2002b). Their Ciona savignyi counterparts were
first searched for in genome sequences that were produced by whole-genome
shotgun sequencing and that are deposited in the Trace archive of NCBI. Based
on the genomic sequences, we amplified cDNAs from gastrula and tailbud cDNA
libraries by PCR.
Nucleotide sequences of both strands were determined using a Big-Dye Terminator Cycle Sequencing Ready Reaction kit and an ABI PRISM 377 DNA sequencer (Perkin Elmer, Norwalk, CT, USA).
Whole-mount in situ hybridization
To determine mRNA distribution in eggs and embryos, RNA probes were
prepared using a DIG RNA-labeling Kit (Roche). Whole-mount in situ
hybridization was performed using digoxigenin-labeled antisense probes as
described previously (Satou and Satoh,
1997).
Morpholino oligonucleotides and synthetic capped mRNAs
In the present study, we used 25-mer morpholino oligonucleotides (hereafter
referred to as `morpholinos'; Gene Tools, LLC) for Cs-Twist-like1
(5'-CTTGATTGTACTCTAGTGATGTCAT-3') and Cs-NoTrlc
(5'-ATTTCTCATTACTTCTGTTGACATG-3'). Synthetic capped mRNAs for
Cs-Twist-like1 and Cs-NoTrlc were synthesized from their
cDNAs cloned into the pBluescript RN3 vector
(Lemaire et al., 1995) using a
Megascript T3 kit (Ambion). To obtain capped mRNA, the concentration of GTP
was lowered to 1.5 mM and the cap analog 7mGpppG was added at 6 mM. For rescue
experiments, synthetic mRNAs were designed to lack the morpholino target
sequence and, therefore, the morpholinos did not recognize these synthetic
mRNAs. We also used a morpholino against Cs-Fgf9/16/20
(Imai et al., 2002a
). After
insemination, fertilized eggs were dechorionated, and microinjected with 15
pmol of morpholinos and/or synthetic capped mRNAs in 30 pl of solution using a
micromanipulator (Narishige Scientific Instrument Laboratory, Tokyo), as
described (Imai et al., 2000
).
Injected eggs were reared at
18°C in MFSW containing 50 µg/ml
streptomycin sulfate. Cleavage of some embryos was arrested at the 110-cell
stage using cytochalasin B, and the embryos were then further cultured until
the control embryos reached the desired stage.
Detection of differentiation markers
The following cell-specific markers were used to assess the differentiation
of embryonic cells, and were detected by whole-mount in situ hybridization:
Cs-MA1, a larval muscle-specific actin gene
(Chiba et al., 1998);
Cs-Epi1, an epidermis-specific gene
(Chiba et al., 1998
);
Cs-fibrinogen-like (Cs-fibrn), a notochord-specific gene
(Imai et al., 2002a
);
Cs-Mech1, a mesenchyme-specific gene
(Imai et al., 2002a
); and
Cs-ETR, a pan-neural marker gene
(Imai et al., 2002a
). The
differentiation of endoderm cells in experimental embryos was monitored by the
histochemical detection of alkaline phosphatase as previously described
(Whittaker and Meedel,
1989
).
Quantitative RT-PCR
Twenty-five embryos at the 110-cell stage were lysed in 200 µl of GTC
solution [4 M guanidinium thiocyanate, 50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 2%
sarkosyl, 1% ß-mercaptoethanol] and total RNA prepared. The RNA was used
for cDNA synthesis with oligo(dT) primers as described previously
(Imai et al., 2000). Real-time
RT-PCR was performed using SYBR Green PCR Master Mix and an ABI prism 7000
(Applied Biosystem). A one-embryo-equivalent quantity of cDNA was used for
each real-time RT-PCR. The cycling conditions were 15 seconds at 95°C and
1 minute at 60°C, according to the supplier's protocol. The experiment was
repeated twice with different batches of embryos. Relative expression values
were calculated by comparison with the level of expression in uninjected
control embryos. Control samples lacking reverse transcriptase in the cDNA
synthesis reaction failed to give specific products in all cases. Dissociation
curves were used to confirm that single specific PCR products were
amplified.
The primers used in the present study were as follows:
Cs-NoTrlc, 5'-CTCGCCACCAAACGATCAAT-3' and 5'-CGCGAATGTTGCCAAGTGT-3'; and
Elongation factor 2 (EF2), 5'-CGCAGTGAAACCGAATGATTC-3' and 5'-CCCGATCTTCAATGTCAATGC-3'.
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RESULTS |
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Three of the Ciona intestinalis bHLH genes were closely related to
one another. A previous phylogenetic analysis failed to predict their
orthologous relationship with known bHLH proteins
(Satou et al., 2003), although
the best-hit proteins by BlastP searches were Twist and Atonal
(E-value>3E-07). We identified two Ciona savignyi cDNA clones for
their orthologs (AB105881 and AB105882). One was 1069 bp in length and encoded
a polypeptide of 318 amino acid residues, and the other was 1211 bp in length
and encoded a polypeptide of 255 amino acid residues. Alignment of the bHLH
regions of these proteins and a phylogenetic analysis using the
neighbor-joining method indicated that the last common ancestor of the two
Ciona species had two Twist and Atonal-like genes, and that one of
them was duplicated in the Ciona intestinalis lineage after the
divergence of the two species (Fig.
1A,B). As described below, because these are most likely to be
orthologous to Twist proteins from their function, these two genes were
renamed as Twist-like1 and Twist-like2 [they were previously
called twist-and-atonal-like genes
(Satou et al., 2003
)].
Ciona savignyi has only one Twist-like1 gene
(Cs-Twist-like1) and one Twist-like2 gene
(Cs-Twist-like2), whereas Ciona intestinalis has two
Twist-like1 genes (Ci-Twist-like1a and
Ci-Twist-like1b) and one Twist-like2 gene
(Ci-Twist-like2).
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The fifth bHLH gene is an ortholog of vertebrate Mist genes
[Ci-Mist (Satou et al.,
2003)]. A cDNA clone for the Ciona savignyi ortholog was
1909 bp long and encoded a polypeptide of 537 amino acid residues (AB105883).
This gene was designated Cs-Mist.
The homeobox gene predominantly expressed in mesenchymal cells is an
ortholog of Hex [Ci-Hex
(Wada et al., 2003)]. A cDNA
clone for the Ciona savignyi ortholog was 2296 bp long and encoded a
polypeptide of 524 amino acid residues (AB105885). This gene was designated
Cs-Hex.
Expression patterns of mesenchyme-specific transcription factors
Expression profiles of these genes were re-examined in Ciona
savignyi embryos. The expression pattern of Cs-Twist-like1 is
shown (Fig. 2B). No maternal
expression of Cs-Twist-like1 was detected by whole-mount in situ
hybridization. An extensive EST analysis of developmentally relevant genes of
Ciona intestinalis did not yield any ESTs for
Ci-Twist-like1a/b in an egg cDNA library
[http://ghost.zool.kyoto-u.ac.jp
(Satou et al., 2002b)],
supporting the notion that there was no maternal expression of this gene. The
first zygotic expression was detected in one pair of the presumptive
mesenchymal cells (B7.7) of the 64-cell embryo. Thereafter,
Cs-Twist-like1 was expressed in the remaining two lineages (B8.5 and
A7.6 lines) of mesenchymal cells by the early gastrula stage. This
Cs-Twist-like1 expression continued until the neurula stage and was
then downregulated so that it was rarely detected in the tailbud embryo.
The expression of Cs-Twist-like2 was not detected in early embryos
up to the early gastrula stage (Fig.
2C); Cs-Twist-like2 expression began in cells of every
mesenchymal lineage at the late gastrula stage (refer to 110-cell stage
arrested embryos in Fig. 7B and
Fig. 9A) and continued strongly
in mesenchymal cells of tailbud embryos. This temporal profile of
Cs-Twist-like2 expression is consistent with the EST data of the
Ciona intestinalis counterpart
[http://ghost.zool.kyoto-u.ac.jp
(Satou et al., 2002b)].
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|
The expression pattern of Cs-Mist is shown in Fig. 2E. No maternal Cs-Mist mRNA was detected. The zygotic expression of Cs-Mist began in cells of the B7.7 line mesenchyme between the late gastrula and early neurula stages, and the expression continued thereafter throughout embryogenesis. No mesenchymal cells other than the B7.7 line cells expressed Cs-Mist. This was confirmed in embryos arrested at the 110-cell stage and cultivated up to the stage corresponding to tailbud embryos. Treatment of 110-cell embryos with cytochalasin B disturbs cytokinesis but not the intrinsic differentiation program, which facilitates identification of the lineage of cells with Cs-Mist expression. The treated embryos showed Cs-Mist expression only in the B7.7 line cells (Fig. 2G).
The expression of Cs-Hex was also zygotic (Fig. 2F). Expression was not detected in embryos at the neurula stage or earlier, but zygotic expression was detected in all the mesenchymal cells of the tailbud embryo. This was confirmed in embryos arrested at the 110-cell stage and cultivated up to the stage corresponding to tailbud embryos (Fig. 2H).
FGF9/16/20 regulates the expression of the mesenchyme-specific
transcription factors
A previous study demonstrated that Cs-Fgf9/16/20 induces
mesenchymal identification in ascidian embryos, because inhibition of the
translation of Cs-Fgf9/16/20 mRNA with a specific morpholino resulted
in failure of the differentiation of mesenchymal cells, and overexpression of
Cs-Fgf9/16/20 mRNA resulted in ectopic differentiation of mesenchymal
cells (Imai et al., 2002a). We
examined here, whether or not the transcription of the five transcription
factor genes identified above is under the control of Cs-FGF9/16/20. For this
purpose, Cs-Fgf9/16/20 morpholino was microinjected into fertilized
eggs of Ciona savignyi. The specificity of this morpholino was
demonstrated in a previous study (Imai et
al., 2002a
). The expression of Cs-Twist-like1, Cs-Twist-like2,
Cs-Mist and Cs-Hex was completely suppressed in the
morpholino-injected embryos (0/22 for Cs-Twist-like1; 0/16 for
Cs-Twist-like2; 0/8 for Cs-Mist and 0/12 for
Cs-Hex), indicating that transcription of the genes for these four
factors is regulated by Cs-FGF9/16/20 (Fig.
3). However, Cs-NoTrlc expression was not downregulated
in most experimental embryos (9/14), suggesting that the expression of this
transcription factor gene is at least partly regulated by factor(s) other than
Cs-FGF9/16/20.
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Cs-Twist-like2 and Cs-Hex are also thought to be under the control of Cs-NoTrlc, because Cs-Twist-like2 and Cs-Hex are under the regulation of Cs-Twist-like1. Injection of the Cs-NoTrlc morpholino resulted in loss of expression of Cs-Twist-like2 in TLCs (0/27; refer to Fig. 7B'). Cs-Mist was not examined in the present study, because it is not expressed in TLCs. These results strongly suggest that Cs-Twist-like1 and Cs-NoTrlc play important roles in determination of the mesenchymal fate. Therefore, we further examined the functions of these two genes as described in the following sections.
Twist-like1 is essential for determination of mesenchymal
fate
Microinjection of the Cs-Twist-like1 morpholino into fertilized
eggs resulted in larvae that appeared morphologically normal
(Fig. 5A,A'). The
differentiation of major tissues was examined with molecular markers at the
tailbud stage. Marker genes for epidermis (Cs-Epi1), nervous system
(Cs-ETR) and notochord (Cs-fibrn) were expressed in
experimental embryos (Fig.
5B'-D') as they are in control embryos
(Fig. 5B-D), suggesting that
the differentiation of these tissues is not disturbed by injection of the
Cs-Twist-like1 morpholino. A marker gene for muscle (Cs-MA1)
was expressed in the tail muscle cells of experimental embryos
(Fig. 5E') as it was in
control embryos (Fig. 5E).
However, in all cases, the anterior boundary of Cs-MA1-positive cells
was extended anteriorly (16/16), suggesting ectopic expression of the muscle
marker gene (arrow in Fig.
5E'). Differentiation of endoderm appeared normal, as
indicated by examination of the histochemical staining of endoderm-specific
alkaline phosphatase (AP) in larvae (Fig.
5F,F'; 13/13). However, the differentiation marker for
mesenchyme (Cs-Mech1) was completely lost in experimental embryos
(Fig. 5G,G'; 0/17),
suggesting an essential role for Twist-like1 in the determination of
mesenchymal fate.
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The specificity of the Cs-Twist-like1 morpholino was confirmed by
a rescue experiment. Co-injection of the morpholino and an in vitro
synthesized Cs-Twist-like1 mRNA with the morpholino recognition
sequence resulted in the loss of expression of Cs-Mech1
(Fig. 6A; 0/11) and
Cs-Twist-like2 (Fig.
6B; 0/10), as had occurred in the embryos injected with the
morpholino only (Fig.
5G', Fig.
4A'). However, co-injection of the morpholino and
Twist-like1 mRNA without the morpholino recognition sequence
(M-Cs-Twist-like1) resulted in the recovery of
Cs-Mech1 (Fig.
6A'; 11/11) and Cs-Twist-like2 expression
(Fig. 6B'; 10/10).
Therefore, these results strongly suggest that this morpholino works as a
specific inhibitor of Cs-Twist-like1 mRNA.
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If Twist-like1 plays a central role in mesenchyme determination after induction by Cs-FGF9/16/20, Twist-like1 overexpression in the Cs-Fgf9/16/20 suppressed embryos should cause recovery of the expression of mesenchyme-specific genes. Microinjection of Twist-like1 mRNA with the Cs-Fgf9/16/20-morpholino into fertilized eggs resulted in recovery of Cs-Mech1 (13/15) and Cs-Twist-like2 (15/15) expression at the tailbud stage (Fig. 5L,M), although the expression was not as strong as that seen when Cs-Twist-like1 mRNA was injected alone. These results suggest that Twist-like1 acts as a `key regulatory gene' in the differentiation of ascidian mesenchymal cells.
NoTrlc is essential for the determination of trunk lateral
mesenchyme cells
Microinjection of the Cs-NoTrlc morpholino into fertilized eggs
resulted in larvae whose morphology appeared normal (data not shown). The
differentiation of major tissues was examined with molecular markers. The
expression of marker genes for epidermis (Cs-Epi1), nervous system
(Cs-ETR), notochord (Csfibrn) and muscle (Cs-MA1)
was normal in the experimental tailbud embryos (data not shown). Normal
differentiation of endoderm was also confirmed by histochemical staining of AP
in larvae (data not shown). However, the differentiation marker of mesenchyme
(Cs-Mech1) was lost in TLCs of the experimental embryos
(Fig. 7A,A'; 0/18),
suggesting an essential role for Cs-NoTrlc in the specification of
TLCs. This was consistent with the loss of Cs-Twist-like2 expression
in TLCs of the NoTrlc morpholino-injected embryos
(Fig. 7B,B'; 0/9 for
tailbud embryos and 0/27 for the 110-cell-arrested embryos). Because
Cs-Twist-like2 is expressed more strongly in TLCs than
Cs-Mech1, we used Cs-Twist-like2 as a marker for TLCs in the
subsequent experiments.
Conversely, overexpression of Cs-NoTrlc by microinjection of its synthetic mRNA led to ectopic expression of Cs-Twist-like2 in experimental tailbud embryos (data not shown; 10/10) and in embryos arrested at the 110-cell stage (Fig. 7C,C'; 27/27), suggesting that Cs-NoTrlc overexpression promotes the ectopic differentiation of mesenchyme.
The specificity of the morpholino was confirmed by a rescue experiment.
Co-injection of the morpholino and in vitro synthesized Cs-NoTrlc
mRNA with the morpholino recognition sequence resulted in the loss of
Cs-Twist-like2 expression in TLCs of experimental tailbud embryos
(Fig. 8A; 0/8) and embryos
arrested at the 110-cell stage (Fig.
8B; 0/16), as it did in embryos injected with the morpholino alone
(Fig. 7B'). However,
co-injection of the morpholino and Cs-NoTrlc mRNA without the
morpholino recognition sequence (M-NoTrlc) resulted in the
recovery of Cs-Twist-like2 expression in the experimental tailbud
embryos (Fig. 8A'; 9/9)
and in the 110-cell arrested embryos (Fig.
8B'; 18/18), suggesting that this morpholino works as a
specific inhibitor of Cs-NoTrlc mRNA.
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Next, the expression of Cs-Twist-like1 and Cs-NoTrlc was examined in Cs-FoxD knockdown embryos, because these genes are the upstream factors of Cs-Twist-like2. Suppression of Cs-FoxD resulted in loss of Cs-Twist-like1 expression in TLCs of early gastrulae (Fig. 9B,B'; 0/31) and of cleavage-arrested embryos (Fig. 9C,C'; 0/14). In the case of Cs-NoTrlc, 74% of experimental embryos lost expression, whereas the remaining 26% expressed Cs-NoTrlc weakly (n=26; data not shown). To confirm this result, quantitative RT-PCR was performed. Template RNA was extracted from 25 embryos and reverse-transcribed. Then real-time PCR was performed using a quantity of cDNA that was equivalent to one embryo. A 56% and 67% reduction of the amount of Cs-NoTrlc mRNA was observed in 64-cell and 110-cell stage embryos, respectively, when compared with uninjected control embryos (Fig. 10). The amount of EF2 mRNA, used as a control, was not changed (Fig. 10). Therefore, Cs-FoxD is highly likely to be one of the upstream factors of Cs-NoTrlc, and some other unknown factors may also regulate Cs-NoTrlc expression.
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DISCUSSION |
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B7.7-line mesenchyme (Fig.
11B)
This line of mesenchyme is probably determined in a similar way to the
B8.5-line. The differences are (1) that Twist-like1 is expressed
earlier in B7.7 than in B8.5, (2) that suppression of Twist-like1
results in ectopic differentiation of muscle cells, and (3) that Mist
is expressed in this line of mesenchyme under the control of
Twist-like1.
A7.6-line mesenchyme (TLCs; Fig.
11C)
Twist-like1 is also essential for the determination of this lineage. As in
the other two lines of mesenchyme, Fgf9/16/20 is required for
expression of Twist-like1 and, thus, differentiation of TLCs. At
present, we do not know when and from where this FGF signal is emitted.
Fgf9/16/20 is also expressed transiently in the presumptive TLC
blastomeres (A5.2 at the 16-cell stage and A6.3 at the 32-cell stage).
Therefore it is also possible that the presumptive TLC blastomeres induce
themselves during this period through an autocrine loop. In addition,
NoTrlc and FoxD are also intrinsically required for
expression of Twist-like1 in this lineage. NoTrlc is also
partially regulated by FGF9/16/20, whereas FoxD is independent of
FGF9/16/20 (Imai et al.,
2002b). Injection of the FoxD morpholino results in a
complete loss of expression of Twist-like1 in this line, although
NoTrlc expression is not completely lost in experimental embryos.
Therefore, NoTrlc and FoxD govern Twist-like1 expression, and
NoTrlc is partially regulated by FoxD. Because FoxD regulation of
NoTrlc is partial, it would appear that there is an unknown factor
that regulates NoTrlc. Kawaminani and Nishida showed by embryological
manipulation of Halocynthia embryos that induction from animal
blastomeres is required for differentiation of TLCs
(Kawaminani and Nishida,
1997
). If this is the case with Ciona, then this signal
might regulate NoTrlc.
In the FoxD-knockdown embryos, the presumptive B-line notochord
blastomeres did not differentiate into notochord
(Imai et al., 2002b) (this
study), but instead expressed Cs-Twist-like1 and
Cs-Twist-like2, suggesting that the B-line notochord precursors
trans-differentiated into the mesenchyme
(Fig. 9A',C').
Because FoxD is essential for notochord
(Imai et al., 2002b
) and TLCs,
the differentiated mesenchyme in the FoxD-knockdown embryos is likely
to be B-line mesenchyme and not TLCs. For example, the B-line notochord
precursors (B8.6) may trace the same developmental fate as their sibling
mesenchymal blastomeres (B8.5) in the FoxD-knockdown embryos.
Nevertheless, it is also possible that the B-line notochord precursors
trans-differentiated into TLCs. At present, we do not have any good marker for
discriminating the B-line mesenchyme from the TLCs; this will be one of the
subjects for future research.
Determination of mesenchyme
In Halocynthia embryos, B-line (B8.5 and B7.7) mesenchyme is
specified or committed to mesenchyme fate at the late 64-cell stage, and TLCs
are specified at the 44-cell stage when A6.3 cleaves into A7.6 (TLC precursor)
and A7.5 (endoderm precursor) (Kawaminani
and Nishida, 1997; Kim and
Nishida, 1999
). The expression of Twist-like1 begins
after the specification of these precursors, and all known mesenchymal genes
in Ciona are completely regulated by Twist-like1. Therefore,
it is likely that Twist-like1 is a transcription factor that determines the
mesenchymal fate, triggering a final step for the differentiation of
mesenchyme.
In Ciona embryos, Cs-FGF9/16/20 and a Notch signaling pathway are
potential inducers of notochord (Corbo et
al., 1998; Imai et al.,
2002a
). After induction, the notochord precursors express
Brachyury, which triggers differentiation of the notochord.
Therefore, a genetic cascade for notochord differentiation converges on
Brachyury, which then activates expression of a variety of
notochord-specific genes. This scheme is probably applicable to mesenchyme.
That is, the genetic cascade for mesenchyme differentiation converges on
Twist-like1.
Conserved and non-conserved mechanisms for mesoderm induction within
chordates
Twist-like1 and Twist-like2 are bHLH factor genes that
have recently duplicated in the ascidian lineage
(Fig. 1B). A Blast search
revealed the factors to have the closest relationship with Twist. However, a
detailed phylogenetic analysis did not support this orthology with a high
bootstrap value, although Twist proteins have been identified in a wide range
of animals from jellyfish to vertebrates. In addition to the bHLH domains,
vertebrate and jellyfish Twist proteins have a conserved motif called the WR
motif (Castanon and Baylies,
2002). The WR motif was not found in Twist-like1 or
Twist-like2. In spite of the lack of supporting evidence for their
orthology, the Ciona intestinalis genome does not contain any genes
closer to Twist than Twist-like1 and Twist-like2
(Satou et al., 2003
).
Therefore, if genes orthologous to Twist have been maintained in the
Ciona genome, Twist-like1 and Twist-like2 are the
most likely candidates.
Twist can be found across various species in cells contributing to mesoderm
and/or its derivatives, suggesting that Twist may have a common role in
mesoderm differentiation (reviewed by
Castanon and Baylies, 2002). In
Drosophila, Twist is required for the induction of mesoderm and
gastrulation, and subsequently acts as an activator in the process of somatic
muscle development. In mice, Twist1 is first uniformly expressed in
the early somites, and is then expressed in the sclerotome and the dermatome,
but not in the myotome, after the somites are compartmentalized. The myotome
expresses other bHLH genes called myogenic factors, including MyoD
and Myf5. Indeed, Twist1 has an inhibitory role in muscle
differentiation.
If Twist-like1 and Twist-like2 are orthologs of
Twist, their roles, especially the role of Twist-like1,
appear to be similar to those of vertebrate Twist. Twist-like1 and
Twist-like2 are not expressed in muscle cells. In ascidian larval
muscle cells, a myogenic factor (AMD1 in Halocynthia and Ci-MDF in
Ciona intestinalis), which represents an ancestral form of four
vertebrate myogenic factors (MyoD, Myf5, myogenin and MRF4), is expressed
(Satoh et al., 1996;
Meedel et al., 1997
). Without
induction by Cs-FGF9/16/20, mesenchyme precursors fail to express
Twist-like1 and differentiate into muscle cells. These ectopic muscle
cells express a myogenic factor (K.S.I., unpublished). This mutually exclusive
expression of Twist-like1 and the myogenic factor is closely
comparable to the vertebrate situation.
In the Ciona intestinalis genome, there are two orthologous genes for Twist-like1 and one orthologous gene for Twist-like2. These three genes are aligned in tandem within 15 kb of the genome. The sequence similarity shown in Fig. 1, and the genomic organization strongly suggests that these genes occurred recently by gene duplications after the divergence of ascidians and vertebrates. The phylogenetic tree shown in Fig. 1B also shows that Twist-like1 was further duplicated in the Ciona intestinalis lineage. Although the function of Twist-like2 has not yet been determined, Twist-like2 may have acquired a novel function during ascidian evolution, if an ancestral function of Twist-like1 and Twist-like2 is the specification of mesoderm (mesenchyme), the common function for Twist among other organisms.
The phylogenetic relationship of another bHLH gene, NoTrlc, with
other known bHLH genes is also not clear. Although a Blast search suggests
that NoTrlc is evolutionarily close to dHand and eHand, the Ciona
genome contains a clear ortholog of vertebrate eHand and
dHand (Satou et al.,
2003). Therefore, NoTrlc characterized in the present
study is not likely to be an ortholog of vertebrate dHand and
eHand. The molecular evolution of the ascidian bHLH genes will be an
intriguing subject for future research.
Intrinsic differences among types of ascidian larval mesenchyme
cells
Ascidian larval mesenchymal cells express many genes for proteins that
function in protein synthesis and RNA metabolism
(Satou et al., 2001;
Kusakabe et al., 2002
). The
mesenchymal cells also have developmental competency as primordia for adult
tissues and organs. Hirano and Nishida examined the developmental fates of
Halocynthia larval mesenchyme cells
(Hirano and Nishida, 1997
).
They reported that B7.7- and B8.5-line mesenchymal cells mainly give rise to
tunic cells, whereas TLCs give rise to blood cells, latitudinal mantle muscle
and ciliary epithelium of gill-slits. In the present study, we identified
three transcription factor genes, Twist-like2, Hex and Mist,
that are expressed during the late embryogenesis. A preliminary experiment
using a morpholino against Twist-like2 did not show any defect in
larvae injected with the morpholino. However, it is possible that these larvae
eventually show defects in the formation of adult organs or tissues after
metamorphosis. Mist is expressed only in the B7.7-line mesenchyme,
suggesting that this line of mesenchymal cells is intrinsically different from
the B8.5-line mesenchyme. Hirano and Nishida reported that, in a few specimens
examined, B7.7-line mesenchymal cells give rise to blood cells and B8.5-line
mesenchymal cells give rise to granules near the holdfast
(Hirano and Nishida, 1997
). If
this is the case with Ciona, the B7.7- and B8.5-line mesenchymes may
have intrinsically different potentials to construct adult organs during
larval development.
Ciona intestinalis has only 16,000 protein-coding genes in
its genome (Dehal et al.,
2002
), and an EST/cDNA project has collected numerous cDNAs of
this animal (Satou et al.,
2002b
). We now have a cDNA collection covering more than 85% of
Ciona genes and more than 90% of developmental genes
(Satou and Satoh, 2003
;
Satoh et al., 2003
). The draft
genome sequence and this cDNA collection open the door to a comprehensive and
integrated analysis of developmental mechanisms at the molecular level. In the
present study, we have shown that comprehensive screening is one of the most
powerful approaches to elucidating the molecular mechanisms of development in
the Ciona system.
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ACKNOWLEDGMENTS |
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