Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
* Author for correspondence (e-mail: kasumi{at}ascidian.zool.kyoto-u.ac.jp)
Accepted 27 November 2003
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SUMMARY |
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Key words: Ciona intestinalis, Notochord, Brachyury, ZicL, Direct activator
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Introduction |
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One of the ß-catenin downstream genes, Fgf9/16/20, is
involved in the induction of mesenchyme by endoderm, while its role in
notochord induction appears partial because Brachyury is expressed in
Fgf9/16/20-suppressed embryos, although its activation is retarded
(Imai et al., 2002a). In
addition, a forkhead transcription factor gene, FoxD, and a zinc
finger transcription factor gene, ZicL, are involved in the process
of notochord specification. The expression of Cs-FoxD, a direct
target of ß-catenin, commences in A5.1, A5.2 and B5.1 at the 16-cell
stage (Fig. 1G), and is
maintained in A6.1, A6.3 and B6.1 at the 32-cell stage
(Fig. 1H) but becomes
undetectable by the 64-cell stage (Fig.
1I). FoxD is not always required for endoderm
differentiation but is essential for notochord differentiation
(Imai et al., 2002b
). However,
ZicL is expressed in A6.2, A6.4, B6.2 and B6.4 at the 32-cell stage
(Fig. 1B,H), and A7.3, A7.4,
A7.7, A7.8, B7.3, B7.4, B7.5, B7.7 and B7.8 at the 64-cell stage
(Fig. 1C,I)
(Imai et al., 2002c
). A6.2,
A6.4, B6.2 and B7.3 are presumptive notochord cells, while A7.3, A7.7 and B8.6
are primordial notochord cells. The expression of Cs-ZicL in the
A-line cells is activated downstream of ß-catenin/FoxD
(Imai et al., 2002b
). The
Cs-ZicL expression is essential for the differentiation of A-line
notochord cells but not of B-line notochord cells
(Imai et al., 2002c
).
Corbo et al. (1997)
investigated the sequence upstream of Ci-Bra to show that the 434-bp
upstream of the TATA box is sufficient as a minimal enhancer for the
notochord-specific expression of a reporter gene (cf. Figs
3,
4). This enhancer contains
recognition sequences of the Suppressor of Hairless [Su(H)], suggesting the
possibility that the Notch signaling pathway plays a role in notochord
differentiation (Corbo et al.,
1998
). Imai et al.
(2002b
) suggested that, in
B-line notochord cells, FoxD activates the Notch signaling pathway, which is
likely to eventually lead to the activation of Brachyury. Therefore,
in B-line cells, ß-catenin
FoxD
Notch
Brachyury is the most probable genetic cascade leading to notochord
differentiation.
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Materials and methods |
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Construction of recombinant plasmids for GST fusion proteins
cDNA fragments encoding the zinc finger motifs (ZF) of ZicL of both C.
intestinalis and C. savignyi were prepared by PCR using the
following primers and cDNAs as templates: Ci-ZicL(ZF),
5'-CGCGGATCCACCAATCCGATTGCTTGC-3' and
5'-CCGCTCGAGGGAACTGTTGTCGATTACG-3', cicl002e04; Cs-ZicL(ZF),
5'-CGCGGATCCTCGCATCCAGTCGCTTG-3' and
5'-CCGCTCGAGTTGTGACGAATTGATGACG-3', BAB68356.
The resultant cDNA fragments were digested with BamHI and XhoI, and inserted into pGEX-4T-1 expression vector (Amersham Pharmacia).
Expression and purification of the proteins
GST fusion proteins as well as GST proteins were prepared using the
DH5 strain of Escherichia coli. Proteins used for binding site
selection analyses were purified from crude bacterial extracts using
glutathione Sepharose 4B according to the manufacturer's instructions
(Amersham Pharmacia). For gel mobility shift assays, proteins were eluted from
the beads in binding buffer (25 mM HEPES (pH 7.5), 100 mM KCl, 10 µM
ZnSO4, 1 mM DTT, 0.1% NP-40, 5% glycerol) containing 10 mM reduced
glutathione. Purified proteins were analyzed by 10% SDS-PAGE.
Binding site selection assay and mutational analyses
DNA sequences optimal for binding with GST/ZicL(ZF) fusion protein were
determined by a PCR-assisted binding site selection assay basically as
described by Pollock and Treisman
(1990). Initially, random
sequence libraries were generated by a primer extension reaction with 61-mers
(5'-GGCCGCTCTAGAACTAGTGGATC(N)16CGATACCGTCGACCTCGAGGG-3')
and 70-mers
(5'-GGCCGCTCTAGACTGCTGTTCG(N)26CGATACCGTCGACCTCGAGGG-3')
as template and R1 primer (5'-CCCTCGAGGTCGACGGTATCG-3') in a
Klenow reaction mixture. Twenty-five picomoles of double-stranded DNA
fragments from the above random sequence libraries were mixed with about 8 ng
of the GST fusion protein or GST protein bound on Glutathione Sepharose 4B
beads (Amersham Pharmacia) in 100 µl of binding buffer consisting of 25 mM
HEPES (pH 7.5), 100 mM KCl, 10 µM ZnSO4, 1 mM DTT, 0.1% NP-40,
5% glycerol and 50 µg/ml poly[dI-dC]. After incubation on ice for 30
minutes, the beads were washed in a washing buffer (25 mM HEPES (pH 7.5), 100
mM KCl, 10 µM ZnSO4, 1 mM DTT, 0.1% NP-40, 5% glycerol) and
treated with proteinase K, and the DNA fragments were purified. A fraction of
the resultant DNA fragments was amplified by PCR with primers (F1:
5'-GGCCGCTCTAGAACTAGTGGATC-3' or F2:
5'-GGCCGCTCTAGACTGCTGTTCG-3' and R1:
5'-CCCTCGAGGTCGACGGTATCG-3'). The PCR products were resolved by
10% polyacrylamide gel electrophoresis, and the bands corresponding to 61- or
70-bp DNA fragments were excised from the gel and eluted into TE buffer.
Purified DNA fragments were subjected to the next round of selection. Ten
rounds of selection were performed, with PCR amplification after each round.
After the 8th and 10th cycles, the PCR products were subcloned into pGEM-T
vector and analyzed.
EMSA
Electrophoretic mobility shift assays (EMSAs) were carried out under the
following conditions. Each reaction contained 50 mM
-32P-labeled substrate DNA fragment and 200 ng of purified
GST fusion protein or GST protein in 20 µl of binding mixture, consisting
of 25 mM HEPES (pH 7.5), 100 mM KCl, 10 µM ZnSO4, 1 mM DTT, 0.1%
NP-40, 5% glycerol, and 50 µg/ml poly[dI-dC]. DNA fragments were prepared
by annealing complementary oligonucleotides, which are shown in
Fig. 2C and
Fig. 3B, for example, ZicL-b:
5'-ACTAGTGGATCCCGCTGTG-3' + 5'-CCGCACAGCGGGATCCACT-3'.
The resultant double-stranded DNA fragments were labeled with
[
-32P]dCTP by Klenow fill-in reaction and purified with a
Microspin S-200 column (Amersham Pharmacia). The binding mixtures were
incubated at room temperature for 30 minutes, and subjected to electrophoresis
on an 8% native polyacrylamide gel. The products were visualized by
autoradiography of the dried gel. For competition reactions, unlabeled DNA
fragments identical to ZicL-b (X100 molar excess) were pre-incubated with the
protein for 10 minutes prior to the addition of labeled probe. In the zinc
removal experiment, 25 mM EDTA was added to the binding mixture.
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Electroporation and microinjection
Different fusion gene constructs were simultaneously electroporated into
fertilized eggs as described (Corbo et al.,
1997). Each electroporation used eggs from several different
batches, and each construct was examined by two or more electroporations. For
microinjection, these constructs were used after linearization with
HindIII. The 25-mer morpholino antisense oligonucleotide for
Ci-ZicL, 5'-GATCAACCATTACATTAGAATACAT-3', was order-made
(GeneTools, LLC). In vitro synthesized capped mRNA of Ci-ZicL was
prepared from cDNA cloned into pBluescript RN3 vector using Megascript T3 kit
(Ambion). Microinjection was performed as described previously
(Imai et al., 2000
;
Imai et al., 2002c
). Solutions
of about 0.5 nM fusion genes, 0.5 mM morpholino and 1.2 µM synthesized mRNA
were used for microinjection, and each injection contained 30 pl of
solution.
Whole-mount in-situ hybridization and histochemical detection of ß-galactosidase activity
Expression of transgenes was visualized by whole-mount in-situ
hybridization (Satou et al.,
2001a) or histochemical detection of ß-galactosidase activity
(Corbo et al., 1997
). For
histochemical detection, embryos were fixed for 15 minutes in 0.5 M NaCl, 0.1
M MOPS (pH 7.5) containing 4% paraformaldehyde, rinsed in PBT and incubated in
PBT containing 1 mM MgCl2, 3 mM K4[Fe(CN)6],
3 mM K3[Fe(CN)6] and 250 µM X-gal.
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Results |
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Characterization of binding sequences of Ci-ZicL
Fusion proteins of GST/Ci-ZicL(ZF) and GST/Cs-ZicL(ZF) were obtained using
pGEX-4T-1, and purified (see Materials and methods). SDS-PAGE
(Fig. 2A) confirmed the
purification of the protein with bands of the expected mobility.
Recognition sequences or binding elements of Ci-ZicL(ZF) and Cs-ZicL(ZF) were identified by PCR-assisted binding site selection assays and EMSAs. Identical results were obtained for both proteins, and therefore we describe here only that for Ci-ZicL(ZF). dsDNAs consisting of the selected 16-bp or 26-bp sequences were examined after five, eight, or 10 rounds of selection. The results for 16-bp and 26-bp dsDNAs appeared similar, although the consensus among the 26-bp dsDNAs was weaker than that among the 16-bp dsDNA (data not shown). Fig. 2B shows the raw numbers of nucleotides obtained after the selection with 16-bp random dsDNAs. Eight and 10 rounds of selection yielded a similar consensus sequence, and this was the case for both GST/Ci-ZicL(ZF) and GST/Cs-ZicL(ZF). This analysis demonstrated that most of the sequences selected had CCGCTGTG at the 3' terminus of the primer F1 (ggatc).
A double-stranded oligonucleotide, `ZicL-b'
(Fig. 2C, upper:
5'-ACTAGTGGATCCCGCTGTGCGG-3') was synthesized. The specific
binding of the fusion protein to the suggested sequence was confirmed by EMSA
with ZicL-b (Fig. 2D, lane 2).
We performed a series of mutation analyses to determine whether a part of the
primer F1 sequence was included in the binding sequence, and confirmed that
CCCGCTGTG was required for effective binding (data not shown). In addition, to
examine the significance of each of the nucleotides for binding, we further
performed EMSAs with various oligonucleotides that had a nucleotide
substitution (AC or T
G transversion) in these sequences. The
results are shown in Fig. 2C,D.
The T to G mutation (Fig. 2C, µA) in the region neighboring the above-mentioned recognition sequence did
not significantly alter the binding affinity to GST/Ci-ZicL(ZF)
(Fig. 2D). Mutations µB,
µC, µD, µI and µJ, in which a nucleotide near one of the ends of
the recognition sequence is substituted, reduced the binding affinity.
Moreover, mutations µE (G to T), µF (C to A), µG (T to G) and µH
(G to T) in the core region of the recognition sequence resulted in loss of
the binding.
Altogether, these findings indicated that the most probable binding sequence of GST/ZicL(ZF) is 5'-CCCGCTGTG-3', and its core, GCTG, is critical for the binding.
The 5' minimal enhancer of Ci-Bra contains two Ci-ZicL-binding elements
Corbo et al., (1997) showed
that the 434 bp upstream of the TATA box (483 bp upstream of the putative
transcription start site) of Ci-Bra is sufficient as a minimal
enhancer for the notochord-specific expression of reporter genes (GFP
and lacZ) (Fig. 3A).
The minimal Ci-Bra enhancer consists of three regions. The most
proximal region (
188) includes three E-boxes and is involved in ectopic
reporter expression in mesenchyme and muscle cells. This region is also
required for expression in notochord cells. The slightly more distal region
between -299 and -188 contains two Su(H) binding motifs and is essential for
activation of Ci-Bra in notochord cells (see also
Corbo et al., 1998
). The distal
region between -434 and -299 includes a Snail binding motif and is associated
with repression of the ectopic expression in mesenchyme and muscle cells (see
also Fujiwara et al., 1998
;
Erives et al., 1998
). In a
report by Corbo et al. (1997
),
the TATA box (shown by a dotted line in
Fig. 3A) was regarded as +1. In
the present study, the 5'-end residue of the Ci-Bra cDNA
sequence that is registered in the database related to the genome sequence was
regarded as +1 of the transcription start site. The TATA box is therefore
located at -44 to -49. In the enhancer there are four putative Snail binding
sites (sna1 to sna4), two putative Su(H) binding sites [Su(H)1 and Su(H)2] and
three E-box sequences (E1 to E3).
We searched the Ci-ZicL-binding element in the 2-kbp region upstream of Ci-Bra and found six elements where more than seven out of the nine nucleotides were identical to the GST/Ci-ZicL(ZF) binding consensus sequence determined as described above. These possible ZicL-binding sequences were located at -123, -168, -731, -1053, -1279 and -1996, and also at -241, which was around the Su(H)2 site, with six out of nine nucleotide identity (Fig. 3B). The -123 sequence, CACAGCTGG (complementary sequence: CCAGCTGTG), and the -168 sequence, CCAGCTGTG, were identical and shared eight out of nine nucleotide identity with the determined binding sequence (underlined); notably, six successive nucleotides, including the core sequence GCTG, were identical. The -123 and -168 elements overlap with E3 and E2, respectively.
We examined whether these sequences were recognized by GST/Ci-ZicL(ZF). As shown in Fig. 3C, the -123 (lane 3) and -168 (lane 4) sequences bound to the fusion protein with a similar affinity as ZicL-b (lane 2), while -731 (lane 6) bound less tightly. Therefore, it is highly likely that Ci-ZicL recognizes the -123 and -168 upstream sequences of Ci-Bra (hereafter designated ZicL-b1 and ZicL-b2, respectively), suggesting the possibility that Ci-ZicL acts as a direct activator of Ci-Bra.
Furthermore, sequences resembling the Ci-ZicL binding element were also present in the upstream region of the presumptive C. savignyi Brachyury gene (Fig. 3D). The upstream sequence of Cs-Bra has been characterized (Y.S. and K. S. Imai, unpublished). Possible ZicL-binding sites were found at -764 and -792 upstream of Cs-Bra. The more proximal one (corresponding to ZicL-b1 of C. intestinalis) had nine out of nine base identity with the binding consensus sequence, while the other had six out of nine base identity. Therefore, ZicL-binding sites, at least ZicL-b1, are likely to function for Cs-Bra expression, too.
The ZicL-binding elements are required for the Ci-Bra enhancer activity
To determine the significance of ZicL-binding sites in the Ci-Bra
enhancer in vivo, a series of Ci-Bra/lacZ fusion genes in which the
Ci-Bra upstream sequence was fused in frame with lacZ with
or without mutations in the potential ZicL-binding sites were introduced into
fertilized eggs. The 3.5-kb genomic DNA fragment upstream from the
Ci-Bra transcription start site contains the cis-regulatory
information required for authentic Ci-Bra expression, since it
mediates a precise, notochord-specific expression pattern of reporter genes in
transgenic embryos (Corbo et al.,
1997), while the 483-bp region appeared to possess `minimal'
enhancer activity. p(-483)Ci-Bra/lacZ, possessing the 483-bp `minimal' or
p(-3.5k)Ci-Bra/lacZ, possessing the 3.5-kbp `full-length' Ci-Bra
enhancer, were mutated at either one or both of the potential ZicL-binding
sites; the ZicL-b1 site, CACAGCTGG [CCAGCTGTG] to
CAgAcgTcG, and the ZicL-b2 site, CCAGCTGTG to CgAcgTcTG.
Using these mutated constructs, we examined the effects of the mutations in
the ZicL-binding sites on the expression of the reporter gene.
When p(-483)Ci-Bra/lacZ or p(-3.5k)Ci-Bra/lacZ was introduced via microinjection and the lacZ expression was assessed by in-situ hybridization, the reporter reproduced the spatial expression pattern of endogenous Ci-Bra at the 64-cell stage (Fig. 4A,D) and at the 110-cell stage (Fig. 4B,E). The reporter expression directed by the 483-bp upstream sequence was weaker. However, when both ZicL-binding sites were mutated, reporter expression was completely abolished (Fig. 4C,F). Introduction of the fusion genes by electroporation gave identical results. Each of these examinations was confirmed with 50 or more embryos. These results indicated that the potential ZicL-binding sites in the Ci-Bra enhancer are essential for the initiation of Ci-Bra transcription.
We also examined the requirement for these ZicL-binding sites at the
tailbud stage, because the Ci-Bra enhancer has been assayed at this
stage (Corbo et al., 1997;
Corbo et al., 1998
;
Fujiwara et al., 1998
) and the
expression of the reporter gene after the prolonged embryogenesis is detected
with better sensitivity even by ß-galactosidase staining. Of 528 embryos
developed from eggs electroporated with p(-483)Ci-Bra/lacZ, 43% showed strong
reporter expression in almost all notochord cells and 26% in a part of the
notochord cells (Fig. 4G,H). In
contrast, as shown in Fig.
4G,I,J, the expression level of p(-483)Ci-Bra/lacZ was markedly
reduced when either ZicL-b1 (123-bp upstream of the transcription start site)
or ZicL-b2 (168-bp upstream) was mutated. In particular, mutation of ZicL-b1
suppressed the expression of the reporter gene, and only 4% of the manipulated
embryos showed partial expression of lacZ
(Fig. 4G,I). In addition, the
ZicL-b1/ZicL-b2 double mutation abolished the reporter expression completely
(Fig. 4G,K).
When p(-3.5k)Ci-Bra/lacZ was electroporated into fertilized eggs, more than
80% of the resultant embryos exhibited strong lacZ expression in almost all
notochord cells at the tailbud stage, as described previously
(Fig. 4G,L)
(Corbo et al., 1997). Mutation
in ZicL-b2 of p(-3.5k)Ci-Bra/lacZ did not cause a significant decrease of the
reporter expression (Fig.
4G,N). However, mutation in ZicL-b1 affected the expression level;
about 53% of embryos still exhibited the reporter expression in most notochord
cells (Fig. 4G), but the
intensity of the expression was clearly weaker than that in controls (compare
Fig. 4M with
Fig. 4L). The proportion of
embryos with weak and partial reporter expression or without expression
increased. Moreover, when both ZicL-b1 and ZicL-b2 were mutated in
p(-3.5k)Ci-Bra/lacZ, the reporter expression was reduced further
(Fig. 4G,O).
Overexpression of Ci-ZicL promotes ectopic expression of Ci-Bra/lacZ reporter
We lastly examined whether or not Ci-ZicL is responsible for
Ci-Bra expression in a manner dependent on these elements in vivo. If
Ci-ZicL binds to these elements to activate the transcription of
Ci-Bra, overexpression of Ci-ZicL by microinjection of its
synthetic mRNA into fertilized eggs should direct ectopic activation of the
reporter gene driven by the Ci-Bra enhancer in blastomeres of
non-notochord lineages, and conversely, suppression of Ci-ZicL should
inactivate the reporter expression.
As shown in Fig. 5A, injection of Ci-ZicL mRNA caused ectopic transactivation of the co-injected reporter gene p(-3.5k)Ci-Bra/lacZ at the 110-cell stage (compared with Fig. 4E). In contrast, p(-3.5k)Ci-Bra/lacZ with double mutation of ZicL-b1 and ZicL-b2 was not activated by co-injection of Ci-ZicL mRNA (Fig. 5B). Moreover, the reporter expression was lost by suppression of endogeneous Ci-ZicL with specific morpholino (Fig. 4C).
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Discussion |
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Corbo et al. (Corbo et al.,
1997) reported that the most proximal region (
188) of the
minimal Ci-Bra enhancer is mainly involved in ectopic expression of a
reporter gene in mesenchyme and muscle cells, while the slightly more distal
region between -299 and -188 (including two Su(H) binding motifs) is essential
for activation of Ci-Bra in notochord cells
(Corbo et al., 1998
), and the
distal region between -434 and -299 (including a Snail binding motif) is
associated with repression of the ectopic expression
(Fujiwara et al., 1998
;
Erives et al., 1998
)
(Fig. 3A). Their analyses,
however, could not definitively determine whether the proximal-most region of
Ci-Bra is associated with enhancement of the gene expression in
notochord cells, although their data demonstrated the reporter expression of
the p(-188)Ci-Bra/lacZ construct in notochord cells
[fig. 5A of Corbo et al.
(Corbo et al., 1997
)]. The
present study substantiates the findings of the study of Corbo et al.
(Corbo et al., 1997
) by
pointing out the significance of the proximal-most region of Ci-Bra
for its transcription in notochord cells
(Fig. 4G-K). Based on the data
obtained from these two studies (Corbo et
al., 1997
) (this study), together with those from other studies
(Corbo et al., 1998
;
Erives et al., 1998
;
Fujiwara et al., 1998
;
Imai et al., 2002c
), we
propose a basic scenario for the transcriptional control of Ci-Bra as
follows (Figs 1,
6).
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One question here is whether FoxD directly activates the transcription of ZicL or not. One possibility is that, as is evident in Fig. 1G,H, although A6.2, A6.4 and B6.2 do not show FoxD expression, they are daughter cells of A5.1, A5.2 and B5.1 with FoxD expression. Therefore, FoxD is expressed in A5.1, A5.2 and B5.1 at the 16-cell stage, and the gene product is inherited by the A6.2, A6.4 and B6.2 daughter cells at the 32-cell stage, where it activates ZicL transcription. Another possibility is that FoxD in A6.1, A6.3 and B6.1 activates genes for inducer molecule(s) for signal transduction to promote ZicL expression in A6.2, A6.4 and B6.2 at the 32-cell stage. Once ZicL is transcribed and translated, the gene product would activate Ci-Bra via ZicL binding elements in the proximal-most region of Ci-Bra. This may be a primary genetic cascade for differentiation of A-line notochord cells (Fig. 6A).
The slightly more distal region between -299 and -188 (including two Su(H)
binding motifs) and the distal region between -434 and -299 (including a Snail
binding motif) are primarily associated with Ci-Bra expression in
B-line notochord cells (Fig.
6B). That is, because Ci-ZicL is also expressed in B-line
mesenchyme and muscle cells (B7.3, B7.4, B7.7 and B7.8), Ci-Bra could
be expressed in these cells. However, the snail gene is expressed in
the same cells with Ci-ZicL expression and Snail represses the
Ci-Bra expression there (Fig.
6C and Fig. 1H,I)
(Fujiwara et al., 1998;
Erives et al., 1998
). Fujiwara
et al. pointed out that binding sites sna1 and sna2, but not sna3 and sna4,
are critical for suppressing the Ci-Bra expression
(Fujiwara et al., 1998
).
In B-line notochord cells of Ciona embryos, the Notch signaling
pathway, which is downstream of FoxD, is likely to be involved in the
activation of Brachyury (Imai et
al., 2002b). That is, only B6.2 or B7.3 receives an inductive
signal (probably Notch signaling pathway) from B6.1 or B7.1 (endodermal cell),
which overcomes the Snail repression to govern Ci-Bra expression in
B7.3 (Fig. 6B and
Fig. 1I). However, because the
ZicL-b1/ZicL-b2 double mutation abolishes the reporter gene expression in
B-line notochord cells, ZicL may also be required for Ci-Bra
expression in B-line notochord cells.
Other factors controlling Ci-Bra transcription
As shown in the present study, double mutation of both ZicL binding
elements in the -483 bp minimal enhancer and -3.5 kbp full-length enhancer of
Ci-Bra completely abolished the reporter gene expression
(Fig. 4C,F,K). However, the
same mutation in the 3.5-kbp enhancer region of Ci-Bra did not
completely abolish the expression, but rather affected the level of the
reporter gene expression when examined at the tailbud stage
(Fig. 4G,O). This suggests that
elements other than the two proximal ZicL-recognition sites are also involved
in regulating the transcriptional activity of Ci-Bra. A possible
ZicL-binding sequence found at -731, although with less intense binding
affinity to the GST/Ci-ZicL(ZF) (Fig.
3B), and/or other cis-element(s) might be involved in
regulating the Ci-Bra transcription, particularly in the late
process.
Factors other than ZicL may also be involved in regulating the
transcriptional activity of Ci-Bra. Previous studies have suggested
that the FGF signal transduction pathway is involved in notochord
specification in Ciona embryos
(Imai et al., 2002a), where
Fgf9/16/20 is expressed in blastomeres involved in notochord
differentiation, and Brachyury expression is partially downregulated
by Fgf9/16/20 suppression. In addition, in early embryos of
Ciona as well as other ascidians, FoxA (forkhead;
HNF-3ß) is likely to play roles in endoderm differentiation and
notochord differentiation (Olsen and
Jeffery, 1997
; Shimauchi et
al., 1997
). Factors other than these two may also be involved in
the transcriptional activation of Ciona Brachyury. Therefore, the
present results may be interpreted as indicating that ZicL is a primary and
essential activator of Ci-Bra, leaving open the possibility that
other factors also play roles in Ci-Bra activation.
Transcriptional control of Brachyury in other animal groups
Cis-regulatory elements for specific expression of
Brachyury have been investigated in another ascidian, H.
roretzi (Takahashi et al.,
1999b). The notochord-specific expression of Hr-Bra
depends on a module between -289 and -250 of the upstream sequence of the
gene. The Halocynthia genome also contains two types of
Zic-related genes, macho-1
(Nishida and Sawada, 2001
) and
zicN (Wada and Saiga,
2002
). HrzicN is expressed in blastomeres of muscle,
notochord, anterior mesenchyme and nerve cord, and the gene seems to be
responsible for the differentiation of these types of cell, and a model in
which HrzicN may activate Hr-Bra together with FGF/BMP signaling
pathway is proposed (Wada and Saiga,
2002
). The results of the present study partially support that
model. However, the Ci-ZicL binding element shown here is not present in this
region of Hr-Bra. Therefore, although the
ZicL-Brachyury network appears to function in
Halocynthia notochord specification, details of the
Halocynthia network remain to be elucidated.
In mouse embryos, Brachyury is expressed in the primitive streak
(non-axial mesoderm) and in the node or notochord (axial mesoderm). The
regulatory sequences required for the expression of Brachyury in the
primitive streak are present within 500 bp upstream of the transcription start
site of this gene (Clements et al.,
1996; Yamaguchi et al.,
1999a
). A part of the Brachyury expression domain in the
primitive streak depends on Wnt3a
(Yamaguchi et al., 1999b
) and
the downstream effectors Lef1 and Tcf1
(Galceran et al., 2001
).
TCF-binding sites have been found in the mouse Brachyury promoter,
and their mutation led to loss of Brachyury expression and Wnt
responsiveness (Arnold et al.,
2000
; Yamaguchi et al.,
1999b
). However, cis-regulatory elements responsible for
the notochord-specific expression of the mouse Brachyury have not
been well characterized.
In Xenopus embryos, Xbra and FGF constitute an
autoregulatory loop (Latinki et
al., 1997
; Casey et al.,
1998
). The transcription of Xbra2, via the region 381 bp
upstream of the transcription start site, is activated by FGF and low
concentrations of activin but is suppressed by high concentrations of activin
(Latinki
et al.,
1997
). This suppression is mediated by paired-type homeobox genes,
goosecoid and Mix.1
(Latinki
et al., 1997
;
Latinki
and Smith,
1999
). The Xbra expression also requires zygotic Wnt
activity (Vonica and Gumbiner,
2002
).
At present, however, no conserved trans-regulatory system has been shown to govern the Brachyury transcription in notochord cells in ascidians and vertebrates.
The complexity of ZicL function
As mentioned above, Ci-ZicL appears to function as a direct activator of
Ci-Bra. However, the expression and functions of Ciona ZicL
are multiple and complex, and ZicL expression does not always lead to
Brachyury activation (Imai et
al., 2002c) [see also Wada and Saiga
(Wada and Saiga, 2002
), for
multiple functions of HrzicN]. First, Cs-ZicL is expressed
not only in the A-line notochord lineage but also in the A-line nerve-cord
lineage and B-line muscle lineage. Although the details of ZicL function in
the specification and subsequent differentiation of nerve cord cells are
obscure, ZicL function in B-line muscle cells appears to be coordinated with
the function of another zinc finger transcription factor gene,
Ci-macho1 (Satou et al.,
2002
). Halocynthia macho-1 appears to have the potential
to promote the entire genetic cascade for muscle cell differentiation
(Nishida and Sawada, 2001
). On
the other hand, the potential of Ci-macho1 to promote the genetic
cascade for muscle cell differentiation appears partial, and requires the
collaborative functioning of ZicL
(Imai et al., 2002c
). In
future studies, it should be determined whether the ZicL binding element is
present in the enhancer region of muscle-specific structural genes and serves
to activate their transcription.
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ACKNOWLEDGMENTS |
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