1 Department of Biological Sciences, Graduate School of Science, Tokyo
Metropolitan University, 1-1 Minamiohsawa, Hachiohji, Tokyo 192-0397,
Japan
2 Head Organizer Project, Vertebrate Body Plan Group, RIKEN Center for
Developmental Biology, 2-2-3 Minatojima Minamimachi, Chuou-Ku, Kobe, Hyougo
650-0047, Japan
3 LGPD, IBDM, Case 907, Campus de Luminy, F-13288 Marseille Cedex 09,
France
* Author for correspondence (e-mail: saiga-hidetoshi{at}c.metro-u.ac.jp)
Accepted 14 January 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: C. intestinalis, H. roretzi, Ascidian, Otx
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The conservation of expression domains between ascidian species, and with
vertebrates, raises the possibility of the conservation of regulatory logics
within the chordate lineage. However, cross-species analysis of the activity
of the regulatory regions of Ci-Hox3 in the mouse suggested lack of
conservation (Locascio et al.,
1999). Even between ascidians, two observations have been made
that suggest a possible divergence of regulatory networks in spite of
strikingly similar embryonic development. First, existing cDNA/EST and genomic
data suggest a very poor sequence conservation between Halocynthia
and Ciona. For example, the coding sequences of Halocynthia
and Ciona Brachyury, a T-box gene specifically expressed in the
notochord of both species, are remarkably different
(Marcellini et al., 2003
).
Second, a previous report has suggested that Brachyury, may be
regulated by very different mechanisms in Halocynthia and
Ciona (Takahashi et al.,
1999
).
To readdress the question of the conservation of the regulatory logic among
ascidians, and with vertebrates, we chose Otx as a model, as it is
one of the most phylogenetically conserved developmental genes.
Otx/otd genes have been isolated from various animal species,
including cnidaria, Drosophila, ascidians and vertebrates
(Bally-Cuif et al., 1995;
Finkelstein and Perrimon,
1991
; Hudson and Lemaire,
2001
; Li et al.,
1994
; Pannese et al.,
1995
; Simeone et al.,
1993
; Smith et al.,
1999
; Wada et al.,
1996
). They have the same expression domain, in the anterior part
of embryos, suggesting the evolutionary conservation of essential roles in the
formation and patterning of anterior embryonic territories. Consistently,
Drosophila, mouse or ascidian embryos mutated or knocked down for
Otx/otd genes exhibit defects in head structures, such as deletion or
differentiation deficiency in the anterior central nervous system (CNS)
(Acampora et al., 1996
;
Acampora et al., 1995
;
Ang et al., 1996
;
Finkelstein and Perrimon,
1991
; Matsuo et al.,
1995
; Satou et al.,
2001
, Wada et al.,
2004
).
In mouse embryos, the expression pattern of Otx genes is complex
and very dynamic. Otx2 is first expressed in the anterior visceral
endoderm, then in the anterior epiblast during gastrulation. Later, both
Otx2 and Otx1 are expressed in the developing fore- and
mid-brain (Simeone et al.,
1993). Although the regulatory sequences driving late expression
of Otx after gastrulation have received attention in both mouse and
the puffer fish (Kimura et al.,
1997
; Kimura-Yoshida et al.,
2004
), very little is known about the transcriptional logic
driving the onset of expression of Otx in the endodermal and neural
lineages in vertebrates.
The detailed expression patterns of ascidian Otx genes have been
characterized (Hudson and Lemaire,
2001; Wada et al.,
1996
) (the Otx gene of Halocynthia roretzi has
been designated Hroth but, hereafter, we rename the gene
Hr-Otx according to the recent agreement at the Urochordate Meeting,
Marseille, 2003). They are reminiscent of the expression pattern of mouse
Otx genes, and are very similar between Ci-Otx and
Hr-Otx. Ascidian Otx genes are first expressed in the
endoderm precursors and the anterior CNS precursor cells prior to
gastrulation. In addition, they are expressed during cleavage stages in
mesodermal cells, including in some muscle and trunk lateral cell precursors.
The anterior nervous system and dorsoanterior epidermal cells also express
Otx during neurula and tailbud stages. Previous reports have shown
that 4 kb of Halocynthia genomic sequences upstream of the
translation initiation sites are sufficient to drive late expression of
Hr-Otx at the tailbud stages
(Oda-Ishii and Saiga, 2003
),
and that a similar region in Ciona recapitulates Ci-Otx
expression during cleavage stages (Bertrand
et al., 2003
). Here, we present a careful comparative analysis of
the regulatory logic of Hr- and Ci-Otx before gastrulation,
which reveals a strong conservation of the regulatory logic between ascidians
in spite of poor sequence conservation in the identified enhancers. Our
results also point to a shared regulatory logic in the endodermal territories
between ascidians and vertebrates.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adult ascidians Ciona intestinalis were obtained from the Station de Biologie Marine in Roscoff.
Preparation of reporter constructs
All reporter constructs were prepared by inserting genomic DNA fragments
isolated from the 5'-upstream region or the first intron of Hr-Otx into
the multicloning site of the pPD46.21 vector, a variant of pPD1.27
(Fire et al., 1990), which
harbors the lacZ gene with a nuclear localization signal. The genomic DNA
fragments without the putative endogenous promoter of Hr-Otx were inserted
into the multicloning site of pMApro reporter, which includes HrMA4a basal
promoter region. The plasmid of pMApro was prepared by inserting the basal
promoter region of HrMA4a, 36 bp of the 5' upstream region and 58 bp of
the 5' UTR, into the BamHI/SmaI sites within pPD46.21. The reporter
construct p5402-1473 was described previously
(Oda-Ishii and Saiga, 2003
).
Constructs of p812-24MA, #1, #2, #3, #4, #5 and #6 were prepared by inserting
PCR products into the SalI/BamHI sites within the pMApro plasmid DNA. To
prepare #3
T-box, #4
Lhx/Fox, #5
T-box, #6
Fox,
#6
Lhx and #6
Lhx/Fox mutations were introduced into the putative
binding sites for the transcription factors in the constructs of #3, #4, #5
and #6 using a Gene EditorTM in vitro site-directed mutagenesis system
(Promega). Constructs of Lhx/Fox-BS and T-box-BS were prepared by inserting
double-stranded synthetic oligonucleotides into the SalI/BamHI sites within
the pMApro plasmid DNA.
Microinjection of reporter constructs into Halocynthia roretzi fertilized eggs and detection of lacZ transcripts by whole-mount in situ hybridization
Microinjection of reporter gene constructs into fertilized eggs and
whole-mount in situ hybridization were carried out as described previously
(Oda-Ishii and Saiga, 2003).
Construct plasmid DNAs of the circular form were dissolved in 1 mM Tris-HCl,
0.1 mM EDTA (pH 8.) at the concentration of 3 to 20 ng/µl, and about 90 pl
was injected into a fertilized egg. For each construct, 20-70 embryos were
injected, cultured, and examined for lacZ transcripts, and at least
two independent experiments were carried out.
Electroporation of reporter constructs into Ciona intestinalis fertilized eggs and detection of lacZ transcripts by whole-mount in situ hybridization
Electroporation of reporter constructs into fertilized eggs of Ciona
intestinalis and whole-mount in situ hybridization were carried out as
described (Bertrand et al.,
2003). For each construct at least 100 electroporated embryos were
screened and experiments were performed at least twice.
Sequence comparison and binding sites prediction
Sequence comparisons between the Ciona and Halocynthia
upstream regions were performed using general alignment programs (local:
DNAsis, Blast 2 sequences, Dot plot; global: ClustalW) or
cis-regulatory-devoted comparison programs (Vista, zPicture, Pipmaker and
Family Relations). Binding site predictions were performed with Matinspector
(GATA and Ets) or TFsearch (Fox), or by searching for published consensus
sequences: T-box (Erives and Levine,
2000) or Lhx (Bridwell et al.,
2001
; Mochizuki et al.,
2000
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
While we have previously shown that the first intron of Ci-Otx has
no enhancer activity in Ciona before the onset of gastrulation, a
reporter construct, 3541, in which 3.5 kb upstream of the first exon
was placed upstream of the lacZ reporter gene
(Fig. 2A), recapitulated the
early expression of Otx in Ciona
(Bertrand et al., 2003). To
test whether the global transcriptional logic driving Otx early
expression in the two species is conserved, we introduced the Ci-lacZ
(3541) construct into the fertilized eggs of Halocynthia
roretzi by microinjection and examined the resulting embryos for
lacZ transcripts at the 64-cell stage
(Fig. 2). This construct
directed lacZ transcription in mostly the same pattern as it does in
Ciona (Bertrand et al.,
2003
) with two exceptions. First, the construct also directed
lacZ transcription in the notochord precursors (A7.3, A7.7), in which
Otx transcription is observed in neither species
(Fig. 2B',C). This may
represent an artefactual expression. Second, lacZ transcription was
also detected in the a7.13 blastomeres, which do not normally express
Ci-Otx (Fig. 2C). In
Halocynthia, however, these blastomeres express Hr-Otx
(Wada et al., 1996
). This
suggests that the expression of endogenous Hr-Otx, but not
Ci-Otx, in the a7.13 is not due to differences in the promoter
regions in these two species, but rather to the way the embryos interpret a
common cis-regulatory logic.
|
Transcription of Hr-Otx during the cleavage stage is regulated by line-specific regulatory modules functionally conserved in Ciona
To dissect regulatory regions directing Hr-Otx transcription, we
prepared various deletion constructs of p5402-1473 and p812-24MA and examined
embryos injected with them for lacZ transcripts at the 64-cell stage.
These constructs are summarized in Fig.
3A. This approach allowed us to identify six regulatory modules of
140-280bp (#1 to #6) that are sufficient to drive expression in subdomains of
Hr-Otx-expressing territories
(Fig. 3,
Fig. 4A-D). Regulatory modules
#1 and #2 acted as enhancers, which, when placed upstream of the HrMA4a basal
promoter, directed transcription in both a- and b-line cells expressing
Hr-Otx (Fig.
3B-C',H). By contrast, regulatory modules #3 and #4, placed
in front of the same basal promoter, directed transcription only in the B- and
A-line cells normally expressing Hr-Otx, respectively
(Fig. 3D-E',H). Regulatory module #5, which included the endogenous Hr-Otx promoter,
directed transcription in the Hr-Otx-expressing cells of the a-, b-
and B-lines, but not in the A-line cells
(Fig. 3F,F',H). Finally,
the intronic regulatory module #6, placed in front of the HrMA4a basal
promoter, directed transcription in the A-line cells expressing
Hr-Otx, as well as some ectopic expression in a-line cells
(Fig. 3G,G',H). These
results suggest that the complex expression pattern of Hr-Otx during
cleavage stage is the result of the combined activities of multiple
transcription regulatory modules with distinct specificities, each, in most
cases, working in one or two lines only.
|
|
|
Putative binding sites for transcription factors in the regulatory modules of Otx
Regulatory modules required for Ci-Otx early expression in
specific lines in Ciona have been identified previously
(Bertrand et al., 2003). The
functional conservation of Hr-Otx line-specific enhancer modules
suggests that the regulatory logic driving Otx expression in each
line is conserved between species without sequence conservation between the
modules of the two species. To further test this hypothesis, we compared the
putative binding sites present in regulatory modules of similar specificities
in both Ciona and Halocynthia.
In Ciona, activation of Otx in the neural a- and b-lines
results from the action of clustered ETS- and GATA-binding sites, which
transduce the neural inducing activity of Ci-Fgf9/16/20
(Bertrand et al., 2003). The
two Hr-Otx regulatory modules capable of directing transcription in
the ab-line cells of both Ciona and Halocynthia (#2 and #5)
contain clustered binding sites for both Ets and GATA factors (#2, eight GATA
sites, one Ets site; #5, four GATA sites, one Ets site; see Fig. S1 in
supplementary material), suggesting conservation of the role of ETS and GATA
factors in these lines.
In the vegetal lines, it has been demonstrated that Ciona savignyi
Otx is positively regulated by ß-catenin
(Satou et al., 2001), and
three additional ß-catenin targets expressed earlier than Otx in
the endodermal lineage have been identified, which are the Lim homeobox gene
Cs-Lhx3 and the winged helix genes Cs-FoxD and
Cs-Hnf3. Similarly, the Halocynthia orthologs of
Cs-Lhx3 (Hr-Lim) and Cs-Hnf3 (Hr-FoxA5)
are expressed in the A-line before Otx
(Wada et al., 1995
)
(Shimauchi et al., 1997
).
Consistent with the possibile involvement of these or similar factors in
Hr-Otx regulation, the A-line modules #4 and #6 shared clustered
putative binding sites (hereafter, a putative binding site will be referred to
as a BS) for Lhx and Fox (#4, six Lhx sites, two Fox sites; #6, four Lhx
sites, one Fox site; see Fig. S1 in supplementary material). In
Ciona, consensus BSs for Lhx and Fox factors were also found in the
module necessary for early A-line expression
(Fig. 6, see also Fig S1 in
supplementary material).
|
As the transcriptional regulatory logic of Otx in the vegetal hemisphere is less well understood than that driving expression in the animal lines, we focused on the transcription factors regulating Hr-Otx transcription in the A- and B-lines, and in particular on the role of the Lhx, Fox and T-box BSs identified above. For this, we next examined the effect of mutating these BSs on the transcriptional activity of the identified enhancers.
Putative Lhx and Fox binding sites have partially overlapping roles in Hr-Otx transcription in A-line cells
We introduced two point mutations into each of the BSs for Lhx and/or Fox
in regulatory modules #4 and #6. Mutation of all Lhx BSs, or of all Fox BSs,
had no significant effect on the activity of module #6
(Fig. 4B). However, combined
mutations of all identified Lhx and Fox BSs (#6L/F) led to inactivation
of the module (Fig. 4B).
Mutation of Lhx and/or Fox BSs in module #4 also led to its inactivation (data
not shown, Fig. 4A). The lower
intrinsic activity of this module (Fig.
4A) may account for its inactivation by single mutations.
Consistent with a conserved regulatory logic between Halocynthia and
Ciona, the activity in Ciona of modules #4 and #6 was also
abolished by mutating all the Lhx and Fox BSs
(Fig. 5G,H).
Next, we tested whether combinations of Lhx and Fox BSs were sufficient to drive expression in the A-line. First, we prepared a reporter construct, Lhx/Fox-BS (Fig. 4E), in which a 30-bp DNA fragment derived from the regulatory module #6 containing two BSs for Lhx immediately downstream of a single BS for Fox was placed upstream of the HrMA4a promoter driving lacZ (Fig. 4E). This construct was active and mainly drove lacZ transcription in the A-line Hr-Otx-expressing cells (Fig. 4E). Likewise, the construct Lhx-BS, containing four Lhx BSs but no Fox BSs in the same pMA-lacZ context, drove lacZ transcription in the A-line. This construct, however, also drove ectopic activation in the B-line (Fig. 4E). Finally, a construct containing four Fox BSs but no Lhx BSs directed lacZ transcription in the Otx-expressing A-line, as well as in the other A-line cells and in the a-line cells (Fig. 4E).
Taken together, the results from this section show that both Lhx and Fox BSs are important for the activity of the regulatory modules #6 and #4 in both Ciona and Halocynthia, suggesting that in both animals, Otx may be a direct target of Lhx and Fox proteins. These transcription factors show some redundancy of function, as a multimer of either Lhx or Fox sites was sufficient to drive A-line expression.
Putative T-box binding sites are required for directing Hr-Otx transcription in the B-line cells
The regulatory modules #3 and #5, capable of directing lacZ
transcription in the B-line cells expressing Hr-Otx
(Fig. 4C,D), contain five and
three T-box BSs, respectively. When all of the T-box BSs were mutated
(#3T, #5
T), both regulatory modules lost their ability to direct
lacZ transcription in the Halocynthia B-line cells
(Fig. 4C,D). This requirement
for T-box BSs extended to Ciona embryos, as mutation of those BSs
reduced #3 expression in Ciona B-line cells
(Fig. 5G). Taken together,
T-box proteins are direct regulators of Otx transcription in the
ascidian B-line cells.
However, a 40-bp DNA fragment, T-box-BS, including four T-box BSs from #3, scarcely directed transcription (Fig. 4E). Therefore, it is likely that T-box proteins collaborate with other factors to direct Hr-Otx transcription in the B-line cells.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Features of the Otx cis-regulatory logic in ascidians
Lessons from other invertebrate and vertebrate systems led to the
formulation of a set of rules guiding the properties of cis-regulatory modules
active in developmental systems (Arnone et
al., 1997; Davidson et al.,
2002
; Kulkarni and Arnosti,
2003
). First, each module acts in a distance-independent manner on
exogenous minimal promoters, and their activity is independent of the other
modules. Second, characterized regulatory modules in flies, mouse and sea
urchin bind, on average, close to five different transcription factors
belonging to different classes. Third, the activity of the modules requires
their binding by both transcriptional activators and repressors. Fourth,
modules are often redundant, the deletion of one causing the expression of the
corresponding gene in its territory of activity
(Arnosti, 2003
). Last, in
contrast to some terminal differentiation enhancers in which the orientation
and spacing of TF-binding sites are important, the arrangement of sites found
in developmental modules seems to be plastic
(Struhl, 2001
). The regulatory
modules identified in this study respect some of these rules, but depart from
others.
As in other systems, the modules described here, have a modular nature and
they are able to activate exogenous minimal promoters when placed immediately
upstream. Hence, the precise distance that separates them from the promoter
does not appear to be critical. Nevertheless, each of the ab, A and bB modules
found in Ciona by deletion analysis correspond to a
Halocynthia module of similar activity located in a similar position
(#1+#2, #4 and #5, respectively; see Fig.
6). Whether there is any constraint on the position of the modules
should be considered in future. In addition to these shared modules, two
additional modules are present in Halocynthia, an upstream B-line
module (#3) and a first intron A-line module (#6). The larger genome size of
Halocynthia (Satoh,
1994) thus correlates with an increased redundancy of modules,
rather than with the use of more complex modules. Comparative analysis of the
cis-regulatory sequences of additional genes with a complex expression pattern
will clarify whether this correlation is a general rule in urochordates.
As expected, each module harbors crucial binding sites for transcription
factors of different classes. The ab-module contains ETS (Ets domain) and GATA
(zinc-finger domain) BSs, the A-module, Lhx (homeodomain) and Fox (winged
helix domain) BSs, the B-module T-box BSs, as well as as yet uncharacterised
collaborating binding sites. The presence of these different classes of
TF-binding sites is in keeping with previous work, but it should be noted that
the ab- and A-modules are extremely simple. Their activity can be accounted
for by the presence of only two types of TF-binding sites in each case, as
shown by the reconstruction of their activity by multimers of isolated binding
sites (this work) (see also Bertrand et
al., 2003). In the A-line, the tetramer of Fox- or Lhx-BS directed
expression in the endodermal A-line, and in the rest of the A- and a-lines
(Fox-BS) or in the B-line (Lhx-BS). As the only common territory of activity
between Lhx-BS and Fox-BS corresponds to the A-line endoderm
(Fig. 4E), we propose that the
restriction of the activity of the A-modules to the endoderm is due to a
synergy between the sites whose activities overlap solely in the A-line
endoderm. A similar logic has been demonstrated in the ab-module, which is
solely activated in territories where the activities of GATA- and ETS-binding
sites overlap (Bertrand et al.,
2003
).
We have no evidence for the involvement of crucial repressors in the ab-line and A-line modules, as the activity of these modules can be reconstituted mainly by the combination of two binding sites mediating activation. This contrasts with the general experience from Drosophila and sea urchin, in which very precise expression patterns, such as the one described here, are the result of interplay between activators and repressors.
Finally, comparison of the sequence of the different regulatory modules
found in Ciona and Halocynthia reveals a great plasticity
within each type of module. Evolutionary plasticity was previously found
within Drosophilids in the eve stripe 2 enhancer, in which conserved
sites are spaced differently between species
(Ludwig et al., 1998). Similar
findings were uncovered in rhabditid nematodes
(Ruvinsky and Ruvkun, 2003
;
Webb et al., 2002
) and sea
urchin (Romano and Wray,
2003
). The plasticity observed in the ascidian Otx
modules is much more extensive, probably reflecting greater evolutionary
distance. The number of crucial sites, their order and relative distances all
vary greatly both between similar modules in the two species, and between
Halocynthia modules of similar activity (#1 and #2, #3 and #5, #4 and
#6). This variation suggests that the regulatory syntax is highly degenerate,
reflecting the lack of evolutionary pressure on these modules. A detailed
analysis of the effect of shuffling the position, distance and orientation of
important binding sites in one module would allow further testing of this
hypothesis. The only possible hint of syntax in our study is that binding
sites frequently repeated in a module (e.g. the GATA sites in the ab-modules,
the Lhx sites in #4, the T-box sites in #3 and #5, and Ciona bB) tend
to be evenly distributed along the module, rather than tightly clustered (see
Fig. 6).
In summary, the major features of the cis-regulatory logic of Otx in ascidians are: (1) the general conservation across a large evolutionary distance of the cis-regulatory logic, based on very simple line-specific regulatory modules involving mainly BSs for transcriptional activators; and (2) a conservation of the position of some of these modules between species, which contrasts with the great plasticity of the arrangement of BSs within individual modules. This degeneracy, combined with the involvement of a few types of crucial TF-binding sites, is sufficient to explain how a conserved regulatory logic can be retained in the absence of detectable sequence conservation in the Otx flanking sequences.
Lessons from the comparative analyses of Otx and Brachyury regulatory sequences
As in the case of Otx, no local conservation was found between the
flanking sequences of the Ciona and Halocynthia Brachyury
genes coding for a T-box transcription factor
(Takahashi et al., 1999).
Although the identified Ciona and Halocynthia modules show a
largely conserved activity in cross-species experiments, the types of crucial
binding sites differed markedly between species. The authors thus proposed
that the regulatory logic of Brachyury may differ in
Halocynthia and Ciona. Comparison between two distantly
related echinoderm, the starfish A. miniata and the sea urchin S.
purpuratus, revealed that although some gene regulatory networks have
persisted unaltered since the Cambrian period, others have extensively
diverged (Hinman et al.,
2003
). The comparative analysis of the Brachyury and
Otx regulatory logics between ascidians suggests that a similar
phenomenon may also have occurred in this phylum. It should be noted, however,
that the Brachyury regulatory modules identified in
Halocynthia and Ciona, in spite of being both located close
to the transcription start site, may fulfil different functions. In
particular, while the onset of activity of the Ciona element mirrors
that of Brachyury as demonstrated by a recent study
(Yagi et al., 2004
), little is
known about the onset of activity of the Halocynthia element. The
presence of a crucial T-box-binding site in this latter element suggests that
it may have a maintenance function via a Brachyury auto-regulatory
loop, rather than an activation function.
Transcription factors involved in the activity of the ab-line-specific transcription regulatory module of ascidian Otx
In Ciona, we have previously shown that Otx is activated
in the animal hemisphere (a- and b-line) by the neural inducer
Ci-Fgf9/16/20. This signal is mediated by the transcription factors
Ci-GATAa and Ci-Ets1/2 via a cluster of GATA- and Ets-binding sites in the
Ci-Otx ab-module [referred to as the a-element in Bertrand et al.
(Bertrand et al., 2003)]. In
Halocynthia, it has also been shown that Hr-Ets, the
ortholog of Ci-Ets1/2, is required for Otx activation in the
a- and b-line (Miya and Nishida,
2003
), and here we have shown that the modules driving
Hr-Otx expression in this line (#1, #2, #5) also contain clusters of
GATA- and Ets-BSs. This suggests that the regulatory logic in this line is
conserved between the two species. In addition, #2 and #5 also drive
expression in a- and b-line cells when tested in Ciona. However, #1
does not drive expression in Ciona, indicating that the syntax of the
module is not entirely degenerate and that it partly differs between the two
species. Given the important sequence divergence of at least one of the
factors binding to the a-module, Ets1/2 (50% amino acid identity between
Halocynthia and Ciona), some co-evolution of the module and
its binding factors is not unexpected.
Candidate transcriptional regulators for ascidian Otx in the A-line cells
We showed that the BSs for Lhx and Fox are crucial for the activity of
A-line-specific transcriptional regulation. In Halocynthia and
Ciona embryos, counterparts of Lhx and Fox have
been identified; Hrlim, a member of Lhx3 group of LIM
homeobox gene in Halocynthia
(Wada et al., 1995), five
Lhx genes in Ciona (Imai
et al., 2004
; Satou et al.,
2001
), Hr-FoxA5, a member of FoxA subclass in
Halocynthia (Shimauchi et al.,
1997
), and more than 20 Fox genes in Ciona
(Di Gregorio et al., 2001
;
Imai et al., 2004
;
Satou et al., 2001
).
Lhx3 and several Fox genes are co-expressed in the endoderm
lineage at the 32- and/or 16-cell stage prior to the onset of Otx
expression in the A-line cells and, therefore, represent good candidates for
the activators of Otx transcription in the A-line cells.
It is known that expression of Cs-Otx and Cs-Lhx3 is
upregulated in all blastomeres by ß-catenin overexpression.
Interestingly, at the 32-cell stage, transcription of Cs-Lhx3 was
already upregulated conspicuously, whereas that of Cs-Otx only
occurred later (Satou et al.,
2001), which further suggests that Lhx3 may act upstream of
Otx in the ascidian A-line.
Candidates for a transcription regulator for ascidian Otx in the B-line cells
T-box protein BSs are required for directing Otx transcription in
the B-line cells. In C. intestinalis, Ci-VegTR, a member of the
Tbx15/18/22 subfamily has been proposed to upregulate gene expression
in B-line cells (Erives and Levine,
2000; Takatori et al.,
2004
). Additionally, three Tbx6 subfamily genes are
expressed in B-line cells (Takatori et
al., 2004
). These genes may contribute to the activation of
Ci-Otx in this line. In Halocynthia, three T-box genes,
As-T (Hr-Bra), As-T2 and As-mT, have been
isolated, their expression patterns described, and their putative functions
estimated through overexpression experiments
(Takada et al., 1998
;
Yasuo and Satoh, 1998
). Among
these, As-mT and As-T2 could contribute to the
transcriptional activation of Hr-Otx in B-line cells because their
transcripts are present in B-line cells at the 32-cell stage
(Takada et al., 1998
;
Yasuo et al., 1996
). The
reporter construct containing only the T-box BSs directed lacZ
transcription in Halocynthia embryos only weakly
(Fig. 4E). In addition, the
module #5
T harboring the mutated T-box BSs retained some activity in
Ciona embryos (Fig.
5G). These observations further support the involvement of
additional transcription factors in the regulation of Otx in the
B-line.
Comparison of regulatory mechanism of Otx genes between ascidians and vertebrates
The results of the present study led to the notion that the transcription
regulatory logic of ascidian Otx, involving GATA-, ETS-, Lhx-, Fox-
and T-box-transcription factors, might have been established in the common
ancestor for ascidians of Pleurogona (e.g. Halocynthia) and
Enterogona (e.g. Ciona), which are the two orders that constitute the
class Ascidiacea, and thus the origin of the logic must be very old. To
determine the extent of the conservation, we compared the regulatory
mechanisms of Otx genes between ascidians and vertebrates.
The present study has suggested that a LIM homeoprotein and a Fox protein
are responsible for regulating Otx transcription in the A-line cells
(anterior mesendoderm precursors) of the pre-gastrula stage embryos.
Interestingly, in mouse embryos, Lhx1 and FoxA are
co-expressed in the anterior visceral endoderm (AVE), in which Otx2
is expressed (Perea-Gomez et al.,
1999). Furthermore, the expression of Otx2 during
gastrulation is completely lost in
FoxA/;Lim1/ mutant
embryos (Perea-Gomez et al.,
1999
), suggesting a possibility that Lhx1 and
FoxA are responsible for regulating Otx2 transcription in
mouse embryos in early development. From these results, we propose a
hypothesis that LIM homeoproteins and FoxA proteins are responsible for
regulating Otx gene transcription in both animals, and the regulatory
mechanisms of Otx gene transcription in the endoderm might have been
conserved during chordate evolution. Consistent with the hypothesis,
regulatory regions responsible for the transcription of Otx2 in the
AVE have been identified (Kimura et al.,
2000
), and further analysis of the regulatory mechanism has
revealed that a certain Fox BS in the regulatory region is crucial for the
transcription in AVE (C. Kimura-Yoshida and I.M., personal communication).
Thus Otx genes offer a unique paradigm to advance our knowledge on
the evolutionary conservation or change in chordate embryonic transcriptional
regulatory networks.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/7/1663/DC1
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acampora, D., Mazan, S., Avantaggiato, V., Barone, P., Tuorto, F., Lallemand, Y., Brulet, P. and Simeone, A. (1996). Epilepsy and brain abnormalities in mice lacking the Otx1 gene. Nat. Genet. 14,218 -222.[CrossRef][Medline]
Acampora, D., Mazan, S., Lallemand, Y., Avantaggiato, V., Maury,
M., Simeone, A. and Brulet, P. (1995). Forebrain and
midbrain regions are deleted in Otx2/ mutants due to a defective
anterior neuroectoderm specification during gastrulation.
Development 121,3279
-3290.
Ang, S. L., Jin, O., Rhinn, M., Daigle, N., Stevenson, L. and
Rossant, J. (1996). A targeted mouse Otx2 mutation leads to
severe defects in gastrulation and formation of axial mesoderm and to deletion
of rostral brain. Development
122,243
-252.
Arnone, M. I., Bogarad, L. D., Collazo, A., Kirchhamer, C. V.,
Cameron, R. A., Rast, J. P., Gregorians, A. and Davidson, E. H.
(1997). Green Fluorescent Protein in the sea urchin: new
experimental approaches to transcriptional regulatory analysis in embryos and
larvae. Development 124,4649
-4659.
Arnosti, D. N. (2003). Analysis and function of transcriptional regulatory elements: insights from Drosophila. Annu. Rev. Entomol. 48,579 -602.[CrossRef][Medline]
Bally-Cuif, L., Gulisano, M., Broccoli, V. and Boncinelli, E. (1995). c-otx2 is expressed in two different phases of gastrulation and is sensitive to retinoic acid treatment in chick embryo. Mech. Dev. 49,49 -63.[CrossRef][Medline]
Bertrand, V., Hudson, C., Caillol, D., Popovici, C. and Lemaire, P. (2003). Neural tissue in ascidian embryos is induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors. Cell 115,615 -627.[CrossRef][Medline]
Bridwell, J. A., Price, J. R., Parker, G. E., McCutchan Schiller, A., Sloop, K. W. and Rhodes, S. J. (2001). Role of the LIM domains in DNA recognition by the Lhx3 neuroendocrine transcription factor. Gene 277,239 -250.[CrossRef][Medline]
Davidson, E. H., Rast, J. P., Oliveri, P., Ransick, A.,
Calestani, C., Yuh, C. H., Minokawa, T., Amore, G., Hinman, V.,
Arenas-Mena, C. et al. (2002). A genomic regulatory network
for development. Science
295,1669
-1678.
Di Gregorio, A., Corbo, J. C. and Levine, M. (2001). The regulation of forkhead/HNF-3beta expression in the Ciona embryo. Dev. Biol. 229, 31-43.[CrossRef][Medline]
Erives, A. and Levine, M. (2000). Characterization of a maternal T-Box gene in Ciona intestinalis. Dev. Biol. 225,169 -178.[CrossRef][Medline]
Finkelstein, R. and Perrimon, N. (1991). The molecular genetics of head development in Drosophila melanogaster. Development 112,899 -912.[Medline]
Fire, A., Harrison, S. W. and Dixon, D. (1990). A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans. Gene 93,189 -198.[CrossRef][Medline]
Hinman, V. F., Nguyen, A. T. and Davidson, E. H. (2003). Expression and function of a starfish Otx ortholog, AmOtx: a conserved role for Otx proteins in endoderm development that predates divergence of the eleutherozoa. Mech. Dev. 120,1165 -1176.[CrossRef][Medline]
Hudson, C. and Lemaire, P. (2001). Induction of anterior neural fates in the ascidian Ciona intestinalis. Mech. Dev. 100,189 -203.[CrossRef][Medline]
Imai, K. S., Hino, K., Yagi, K., Satoh, N. and Satou, Y.
(2004). Gene expression profiles of transcription factors and
signaling molecules in the ascidian embryo: towards a comprehensive
understanding of gene networks. Development
131,4047
-4058.
Kimura, C., Takeda, N., Suzuki, M., Oshimura, M., Aizawa, S.
and Matsuo, I. (1997). Cis-acting elements conserved
between mouse and pufferfish Otx2 genes govern the expression in mesencephalic
neural crest cells. Development
124,3929
-3941.
Kimura, C., Yoshinaga, K., Tian, E., Suzuki, M., Aizawa, S. and Matsuo, I. (2000). Visceral endoderm mediates forebrain development by suppressing posteriorizing signals. Dev. Biol. 225,304 -321.[CrossRef][Medline]
Kimura-Yoshida, C., Kitajima, K., Oda-Ishii, I., Tian, E.,
Suzuki, M., Yamamoto, M., Suzuki, T., Kobayashi, M., Aizawa, S. and
Matsuo, I. (2004). Characterization of the pufferfish Otx2
cis-regulators reveals evolutionarily conserved genetic mechanisms for
vertebrate head specification. Development
131, 57-71.
Kulkarni, M. M. and Arnosti, D. N. (2003).
Information display by transcriptional enhancers.
Development 130,6569
-6575.
Li, Y., Allende, M. L., Finkelstein, R. and Weinberg, E. S. (1994). Expression of two zebrafish orthodenticle-related genes in the embryonic brain. Mech. Dev. 48,229 -244.[CrossRef][Medline]
Locascio, A., Aniello, F., Amoroso, A., Manzanares, M.,
Krumlauf, R. and Branno, M. (1999). Patterning the
ascidian nervous system: structure, expression and transgenic analysis of the
CiHox3 gene. Development
126,4737
-4748.
Ludwig, M. Z., Patel, N. H. and Kreitman, M.
(1998). Functional analysis of eve stripe 2 enhancer evolution in
Drosophila: rules governing conservation and change.
Development 125,949
-958.
Marcellini, S., Technau, U., Smith, J. C. and Lemaire, P. (2003). Evolution of Brachyury proteins: identification of a novel regulatory domain conserved within Bilateria. Dev. Biol. 260,352 -361.[CrossRef][Medline]
Matsuo, I., Kuratani, S., Kimura, C., Takeda, N. and Aizawa, S. (1995). Mouse Otx2 functions in the formation and patterning of rostral head. Genes Dev. 9,2646 -2658.[Abstract]
Mitani, Y., Takahashi, H. and Satoh, N. (1999). An ascidian T-box gene As-T2 is related to the Tbx6 subfamily and is associated with embryonic muscle cell differentiation. Dev. Dyn. 215,62 -68.[CrossRef][Medline]
Miya, T. and Nishida, H. (2003). An Ets transcription factor, HrEts, is target of FGF signaling and involved in induction of notochord, mesenchyme, and brain in ascidian embryos. Dev. Biol. 261,25 -38.[CrossRef][Medline]
Mochizuki, T., Karavanov, A. A., Curtiss, P. E., Ault, K. T., Sugimoto, N., Watabe, T., Shiokawa, K., Jamrich, M., Cho, K. W., Dawid, I. B. et al. (2000). Xlim-1 and LIM domain binding protein 1 cooperate with various transcription factors in the regulation of the goosecoid promoter. Dev. Biol. 224,470 -485.[CrossRef][Medline]
Oda-Ishii, I. and Saiga, H. (2003). Genomic organization and promoter and transcription regulatory regions for the expression in the anterior brain (sensory vesicle) of Hroth, the otx homologue of the ascidian, Halocynthia roretzi. Dev. Dyn. 227,104 -113.[CrossRef][Medline]
Pannese, M., Polo, C., Andreazzoli, M., Vignali, R., Kablar, B.,
Barsacchi, G. and Boncinelli, E. (1995). The Xenopus
homologue of Otx2 is a maternal homeobox gene that demarcates and specifies
anterior body regions. Development
121,707
-720.
Perea-Gomez, A., Shawlot, W., Sasaki, H., Behringer, R. R. and
Ang, S. (1999). HNF3beta and Lim1 interact in the visceral
endoderm to regulate primitive streak formation and anterior-posterior
polarity in the mouse embryo. Development
126,4499
-4511.
Romano, L. A. and Wray, G. A. (2003).
Conservation of Endo16 expression in sea urchins despite evolutionary
divergence in both cis and trans-acting components of transcriptional
regulation. Development
130,4187
-4199.
Ruvinsky, I. and Ruvkun, G. (2003). Functional
tests of enhancer conservation between distantly related species.
Development 130,5133
-5142.
Satoh, N. (1994). Developmental biology of ascidians. New York: Cambridge University Press.
Satoh, N., Makabe, W. K., Katsuyama, Y., Wada, S. and Saiga, H. (1996). The ascidian embryo: an experimental system for studying genetic circuitry for embryonic cell specification and morphogenesis. Dev. Growth Differ. 38,325 -340.[CrossRef]
Satoh, N., Satou, Y., Davidson, B. and Levine, M. (2003). Ciona intestinalis: an emerging model for whole-genome analyses. Trends Genet. 19,376 -381.[CrossRef][Medline]
Satou, Y., Imai, K. S. and Satoh, N. (2001). Early embryonic expression of a LIM-homeobox gene Cs-lhx3 is downstream of beta-catenin and responsible for the endoderm differentiation in Ciona savignyi embryos. Development 128,3559 -3570.[Medline]
Shimauchi, Y., Yasuo, H. and Satoh, N. (1997). Autonomy of ascidian fork head/HNF-3 gene expression. Mech. Dev. 69,143 -154.[CrossRef][Medline]
Simeone, A., Acampora, D., Mallamaci, A., Stornaiuolo, A., D'Apice, M. R., Nigro, V. and Boncinelli, E. (1993). A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo. EMBO J. 12,2735 -2747.[Abstract]
Smith, K. M., Gee, L., Blitz, I. L. and Bode, H. R. (1999). CnOtx, a member of the Otx gene family, has a role in cell movement in hydra. Dev. Biol. 212,392 -404.[CrossRef][Medline]
Struhl, K. (2001). Gene regulation. A paradigm
for precision. Science
293,1054
-1055.
Takada, N., Tagawa, K., Takahashi, H. and Satoh, N. (1998). Characterization of an ascidian maternal T-box gene, As-mT. Int. J. Dev. Biol. 42,1093 -1100.[Medline]
Takahashi, H., Mitani, Y., Satoh, G. and Satoh, N.
(1999). Evolutionary alterations of the minimal promoter for
notochord-specific Brachyury expression in ascidian embryos.
Development 126,3725
-3734.
Takatori, N., Hotta, K., Mochizuki, Y., Satoh, G., Mitani, Y., Satoh, N., Satou, Y. and Takahashi, H. (2004). T-box genes in the ascidian Ciona intestinalis: characterization of cDNAs and spatial expression. Dev. Dyn. 230,743 -753.[CrossRef][Medline]
Wada, H., Saiga, H., Satoh, N. and Holland, P. W.
(1998). Tripartite organization of the ancestral chordate brain
and the antiquity of placodes: insights from ascidian Pax-2/5/8, Hox and Otx
genes. Development 125,1113
-1122.
Wada, S., Katsuyama, Y., Yasugi, S. and Saiga, H. (1995). Spatially and temporally regulated expression of the LIM class homeobox gene Hrlim suggests multiple distinct functions in development of the ascidian, Halocynthia roretzi. Mech. Dev. 51,115 -126.[CrossRef][Medline]
Wada, S., Katsuyama, Y., Sato, Y., Itoh, C. and Saiga, H. (1996). Hroth an orthodenticle-related homeobox gene of the ascidian, Halocynthia roretzi: its expression and putative roles in the axis formation during embryogenesis. Mech. Dev. 60, 59-71.[CrossRef][Medline]
Wada, S., Sudo, N. and Saiga, H. (2004). Roles of Hroth, the ascidian otx gene, in the differentiation of the brain (sensory vesicle) and anterior trunk epidermis in the larval development of Halocynthia roretzi. Mech. Dev. 121,463 -474.[CrossRef][Medline]
Webb, C. T., Shabalina, S. A., Ogurtsov, A. Y. and Kondrashov,
A. S. (2002). Analysis of similarity within 142 pairs of
orthologous intergenic regions of Caenorhabditis elegans and Caenorhabditis
briggsae. Nucl. Acids Res.
30,1233
-1239.
Yagi, K., Satou, Y. and Satoh, N. (2004). A
zinc finger transcription factor, ZicL, is a direct activator of Brachyury in
the notochord specification of Ciona intestinalis.
Development 131,1279
-1288.
Yasuo, H. and Satoh, N. (1998). Conservation of the developmental role of Brachyury in notochord formation in a urochordate, the ascidian Halocynthia roretzi. Dev. Biol. 200,158 -170.[CrossRef][Medline]
Yasuo, H., Kobayashi, M., Shimauchi, Y. and Satoh, N. (1996). The ascidian genome contains another T-domain gene that is expressed in differentiating muscle and the tip of the tail of the embryo. Dev. Biol. 180,773 -779.[CrossRef][Medline]