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 18 May 2004
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SUMMARY |
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Key words: Ascidian, Ciona intestinalis, Transcription factors, Signal transduction molecules, Expression profiles
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Introduction |
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Reflecting the simplicity of the larval body plan, ascidian embryogenesis
is comparatively simple. In the egg, maternal determinants for muscle,
endoderm and epidermis are stored, and these tissues differentiate
autonomously. However, inductive signals are required for differentiation of
the notochord, mesenchyme and nervous system
(Nishida, 2002). The molecular
nature of inductive signals has been revealed using embryos of Halocynthia
roretzi, Ciona intestinalis and Ciona savignyi. Although most of
the developmental system is thought to be common between Halocynthia
and Ciona, recent studies have also shown several differences between
them. In fact, they are evolutionarily distant, because Halocynthia
is an Enterogona ascidian and Ciona is a Pleurogona ascidian
these two being major orders of ascidians. Therefore, we describe here mostly
the Ciona system in order to avoid any possible confusion.
In ascidian embryos, the developmental fate of each blastomere is
restricted to one tissue at or before the 110-cell stage
(Fig. 1). In accordance with
this early cell fate restriction, key genes that are essential and sufficient
for differentiation of each tissue are thought to be expressed in each lineage
until this stage. In fact, recent studies have identified such genes for
larval endomesodermal tissues: Lhx3 for endoderm
(Satou et al., 2001a),
Twist-like-1 for mesenchyme, including TLCs
(Imai et al., 2003
),
Brachyury for notochord (Yasuo
and Satoh, 1993
; Yasuo and
Satoh, 1998
; Corbo et al.,
1997a
), and Mesp for TVCs
(Satou et al., 2004
). Muscle
cells are autonomously differentiated by maternally supplied macho-1
encoding a Zic-like transcription factor in Halocynthia
(Nishida and Sawada, 2001
),
and its Ciona homolog is also important for the differentiation of
muscle cells, although an additional mechanism will also be required in
Ciona embryos (Satou et al.,
2002a
; Imai et al.,
2002a
). All the differentiation processes in each lineage are
accomplished through expression of these key genes. Therefore, the
investigation of how these genes are expressed in each lineage will elucidate
specification mechanisms of the ascidian endomesodermal tissues, and
identification of genes downstream of the key genes will lead to understanding
of the differentiation process of each tissue.
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Downstream genes of several of these key genes have been identified. For
example, more than 20 genes downstream of Brachyury have been
reported (Takahashi et al.,
1999; Hotta et al.,
2000
). Among these, genes required for the function of notochord
itself [such as Ci-trop, which encodes tropomyosin-like protein
(Di Gregorio and Levine,
1999
), and Ci-ß4Gal-T, which encodes
ß4-gal-transferase] are included. Genes that may be involved in
morphogenesis of the tail, such as Ci-pk1, which encodes a homolog of
Drosophila prickle, were also identified. Therefore, to understand
the molecular mechanism of the ascidian development, it is important to
analyze the downstream and upstream genes regulated by these key genes.
Although analyses of individual genes have clarified the gene network
behind the endomesoderm differentiation of the ascidian embryo, analyses
focusing on individual genes are insufficient for elucidating the entire
mechanism, because such analyses cannot cover the entire gene repertoire. To
overcome this problem, genome-scale analyses are required. In this respect,
ascidians also provide an ideal experimental system, because the draft genome
sequence of Ciona intestinalis reveals that its genome contains the
basic ancestral complement of genes involved in cell signaling and development
(Dehal et al., 2002). The
ascidian developmental genes are therefore less redundant compared with those
of vertebrates, in which paralogous genes are frequently present, rendering
analyses of gene function difficult.
Towards the comprehensive analysis of Ciona genes, a microarray
covering almost the entire gene repertoire in the Ciona genome has
been published (Azumi et al.,
2003). Quantitative data can be successfully obtained by analyses
using microarrays. However, for studying the development, it is also important
to obtain qualitative data without basic information about where
individual genes are expressed, quantitative data cannot be effectively
evaluated. Based on this line of research, we have already described the
expression profiles of
1000 genes in five developmental stages of
fertilized eggs (Nishikata et al.,
2001
), cleaving embryos
(Fujiwara et al., 2002
),
tailbud embryos (Satou et al.,
2001b
), larvae (Kusakabe et
al., 2002
) and young adults
(Ogasawara et al., 2002
).
The importance of transcriptional regulation of genes in animal development
is obvious (e.g. Davidson et al.,
2002; Levine and Tjian,
2003
). In general, transcription factors can trans-activate other
transcription factor genes and sometimes themselves, making a network of
transcription factor genes. These transcription factor networks are
interlinked with one another and regulated by signaling molecules, such as
Wnt, TGFß and FGF. These networks make a complex body from a single cell
of the fertilized egg and have been partially delineated in ascidian embryos,
as described above. To expand this type of analysis to a genomewide scale and
to understand ascidian development completely, the reconstruction of gene
networks consisting of transcription factors and signaling genes is a good
starting point. Therefore, we address the expression profiles of all
transcription factor genes and major signaling genes. The Ciona
system is ideal for this type of analysis, because the genome contains a
limited number of transcription factor genes and signaling genes compared with
vertebrate genomes. Another advantage is that the expression pattern of each
gene can be described at the single-cell level at and before the 110-cell
stage. To date, expression patterns of several transcription factor genes and
signaling genes have been described, including Brachyury
[Ci-Bra (Corbo et al.,
1997a
)], FoxA-a [Ci-fkh
(Corbo et al., 1997b
)],
Otx (Hudson and Lemaire,
2001
; Satou et al.,
2001a
), Gsx (Hudson
and Lemaire, 2001
), MyoD [Ci-MDF
(Meedel et al., 1997
)],
snail (Corbo et al.,
1997b
; Fujiwara et al.,
1998
), TTF1
(Ristoratore et al., 1999
;
Satou et al., 2001a
),
Dll-A, Dll-B and Dll-C
(Caracciolo et al., 2000
),
Lhx3 (Satou et al.,
2001a
), ZicL (Imai et
al., 2002a
), FoxD
(Imai et al., 2002b
),
macho-1 (Satou et al.,
2002a
), engrailed
(Jiang and Smith, 2002
;
Imai et al., 2002d
), Fgf genes
(Imai et al., 2002c
;
Imai et al., 2002d
),
lefty/antivin (Imai,
2003
), and hedgehog1 and hedgehog2
(Takatori et al., 2002
).
However, these represent only a small part of all the transcription factor and
major signaling genes.
Once the complete gene expression profiles of such genes are described, a variety of genome-scale analyses can be more easily performed, because the number of genes that should be examined becomes limited. In addition, because the function of individual genes can be predicted from their expression profiles, the description of the gene expression profiles also promotes such analyses.
No systematic description of the transcription factor gene expression profiles during embryogenesis has been reported yet in any animal to our knowledge. Therefore, the following description and discussion will also address three fundamental questions. (1) How many and what kind of transcription factor genes are encoded in the Ciona genome? (2) How many such genes are maternally or zygotically expressed? (3) Are there any specific tendencies or differences in the expression patterns between transcription factor families?
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Materials and methods |
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Gene lists
The list for transcription factor genes was obtained by a combination of
three methods. First, genes for basic helix-loop-helix (bHLH), homeodomain
(HD), Fox, ETS, bZIP, nuclear receptor (NR), HMG, T-box transcription factors
or specific categories of zinc-finger transcription factors encoded in the
Ciona genome were previously annotated comprehensively
(Satou et al., 2003a;
Wada et al., 2003
;
Yagi et al., 2003
;
Yamada et al., 2003
). Because
nucleotide sequences of several genes that were recently duplicated are almost
identical, we treated these gene groups as single genes in this study
(Twist-like1a/Twist-like1b, Tbx6b/Tbx6c/Tbx6d,
FoxD-a/FoxD-b and FoxN1/4-a/FoxN1/4-b).
Second, results of Interpro searches of the Ciona proteome were also
used. Ciona proteins with an Interpro domain categorized into
transcription factors by gene ontology terms (GO:3700 or its children) were
added to the list. Finally, we performed homology searches of the
Ciona proteins against the human proteome (IPI, 2003.8.6 version).
When the best-hit human protein of each Ciona protein was annotated
as a transcription factor, the corresponding gene was added to the list.
General transcription factors such as TFIIA were excluded manually from the
list. As shown in Table 1 and
Table S1 (at
http://dev.biologists.org/supplemental),
the list includes 394 transcription factor genes. Known transcription factors
genes that are not included in this list include some zinc-finger genes and
genes that do not have any recognizable motifs or homologs identified as
transcription factors in other animals. As for zinc-finger genes, genes for
nuclear receptors, Zic-related genes and genes encoding homologs that are
identified as transcription factors in humans are all included. Zinc-finger
motifs themselves do not always indicate that a gene encodes a transcription
factor, because even proteins with a C2H2 motif, which is one of the best
known zinc-finger motifs, are not necessarily transcription factors.
Therefore, of 600 zinc-finger genes in the Ciona intestinalis genome
(L. Yamada and N. Satoh, unpublished), only 86 genes that certainly encode a
transcription factor are included in the current list.
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|
All the DIG-RNA probes were synthesized by in vitro transcription with T7
RNA polymerase. The detailed procedure has been described previously
(Satou and Satoh, 1997).
Relative quantification of mRNA by RT-PCR using real-time PCR method
In the present study, we used 25-mer morpholino oligos (referred to as
`morpholinos'; Gene Tools, LLC) for Ci-ß-catenin
(5'-CTGTTCATCATCATTTCAGCCATGC-3'), Ci-Fgf9/16/20
(5'-CATAGACATTTTCAGTATGGAAGGC-3') and Ci-FoxD-a/b
(5'-GCACACAACACTGCACTGTCATCAT-3'). After insemination, fertilized
eggs were dechorionated and microinjected with 15 pmole of morpholinos and/or
synthetic capped mRNAs in 30 pl of solution using a micromanipulator
(Narishige Science Instrument Laboratory, Tokyo) as described
(Imai et al., 2000). Injected
eggs were reared at about 18°C in MFSW containing 50 mg/l streptomycin
sulfate. Embryos injected with these morpholinos exhibited a same phenotype as
Ciona savignyi embryos in which these gene functions were inhibited
with specific morpholinos (Imai et al.,
2000
; Imai et al.,
2002b
; Imai et al.,
2002c
).
Relative quantifications of mRNA were performed by the real-time PCR method
with SYBR-green chemistry using an ABI prism 7000 (Applied Biosystems). The
amplification of a specific product in each reaction was confirmed by
determining a dissociation curve. When the dissociation curve indicated
multiple PCR products, the primer pairs were redesigned until a specific
amplification could be achieved. Primers used in this study are shown in Table
S3
(http://dev.biologists.org/supplemental)
and the detailed procedure has been described previously
(Imai, 2003). Only genes
exhibiting more than threefold difference in the amount of mRNAs by comparison
between morpholino-injected embryos and control embryos were counted as
positive ones. The results for the positive genes were further confirmed by
another independent experiment.
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Results and discussion |
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In the 389 transcription factor genes, only zinc-finger genes that are
readily annotated as a transcription factor gene are included. The
Ciona genome contains 514 additional zinc-finger genes (L. Yamada and
N.S., unpublished). Because the zinc-finger motif itself cannot be used as
evidence that a gene encodes a transcription factor, we cannot know exactly
how many transcription factors are included among the 514 zinc-finger
proteins. Therefore, the number of transcription factor genes encoded in the
Ciona genome would range from 389 to 903 (389+514). The nematode
genome contains about 500 transcription factor genes, about one-third of which
encode a zinc-finger protein (Ruvkun and
Hobert, 1998). The fly genome contains about 700 transcription
factor genes, about half of which encode a zinc-finger protein
(Adams et al., 2000
). However,
the human genome contains about 3000 transcription factor genes
(Lander et al., 2001
).
Therefore, the Ciona genome appears to contain a comparable number of
transcription factor genes to genomes of other invertebrates. This is
consistent with our previous comprehensive annotations of specific families of
transcription factor genes in the Ciona intestinalis genome
(Satou and Satoh, 2003
). For
example, 46 bHLH genes are encoded in the Ciona genome, 38 in the
nematode genome and 58 in the fly genome, but about 130 in the human genome
(Satou et al., 2003a
).
Ninety-two HD transcription factor genes are encoded in the Ciona
genome, 101 in the nematode genome and 113 in the fly genome, but about 270 in
the human genome (Wada et al.,
2003
). Therefore, our list appears to cover almost all
transcription factor genes in the Ciona intestinalis genome.
As shown in the previous studies (Satou
et al., 2003a; Wada et al.,
2003
; Yagi et al.,
2003
; Yamada et al.,
2003
), the Ciona genome does not have any orthologs of
several transcription factor genes found in the vertebrate genome (Table S4).
For example, among bHLH genes, no clear orthologs for NeuroD, Beta3,
Oligo, SCL, NSCL, SRC, Bmal and Clock were found in the
Ciona genome. Some of these genes were lost in the Ciona
genome and the others were probably acquired in the course of vertebrate
evolution after the divergence of ascidians and vertebrates. Therefore, these
missing orthologs were not examined in this study.
The expression profiles of the transcription factor genes
Of 389 genes, cDNA clones for 324 genes were found in our EST collection
(Table 1)
(Satou et al., 2002b;
Satoh et al., 2003
). We tried
to amplify cDNA fragments for the remaining 65 genes from cDNA libraries
derived from eggs, cleaving embryos, gastrulae, tailbud embryos, larvae and
juveniles to obtain cDNA fragments of 28 genes
(Table 1). In other words, we
could not amplify cDNAs for 37 genes in spite of using perfect-match primer
pairs. Our EST collection contains 480,000 ESTs, and 225,238 of these entries
were obtained from cDNA libraries from eggs to tailbud embryos
(Satou et al., 2002b
). Because
such extensive EST analyses and PCR amplifications of cDNAs failed to show
evidence of the gene expression, most of these genes are unlikely to be
expressed during embryogenesis. It is also possible that some of these genes
are pseudogenes or ones mispredicted by gene-prediction programs. The
following analysis was therefore performed for 352 (324+28) transcription
factor genes, and their embryonic expression profiles were examined by
whole-mount in situ hybridization.
In situ hybridization was carried out with fertilized eggs, 16-cell
embryos, 32-cell embryos, 64-cell embryos, early gastrulae (110180 cell
embryos), late gastrulae, neurulae, early tailbud embryos and mid-late tailbud
embryos. The presence or absence of maternal transcripts was classified into
three categories as shown in Table
3. Zygotic expression up to the 64-cell stage was described at the
single-cell level according to Conklin's nomenclature
(Conklin, 1905
). Zygotic
expression at the early gastrula was also described similarly, but names of
blastomeres at the 110-cell stage were used even when the expression after the
110-cell stage was described. This is because cell divisions after the
110-cell stage are rapid and non-synchronous, which makes a large scale
precise sampling of 110-cell embryos difficult. Therefore, for example, when
the present assay says that a given gene is expressed in B7.1 at the early
gastrula stage, this gene is expressed in B7.1 and/or its descendants in the
embryo between the 110-cell stage and the 180-cell stage. Zygotic expression
after the early gastrula stage was described at the tissue level
(Table 3). Trunk ventral cells
(TVCs) and trunk lateral cells (TLCs) have been traditionally regarded as a
part of mesenchyme and it is often difficult to distinguish these cells from
B7.7- and B8.5-derived mesenchyme at the tailbud stage, especially when all of
these lines of the mesenchyme were stained. Therefore, we described TVCs and
TLCs as mesenchyme, although we also describe which mesenchyme expresses a
particular gene as long as we can distinguish between the mesenchymes (this
can be seen at
http://ghost.zool.kyoto-u.ac.jp/tfst.html).
Zygotic transcripts in ascidian embryos can be easily discriminated from
maternal transcripts, because the first zygotic expression signals are
observed in the nucleus.
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|
Maternally expressed transcription factor genes
How many transcription factor genes are maternally expressed in ascidian
eggs? A result of our analysis is shown in Table S5. Because detection of
maternal messages by in situ hybridization is possibly difficult, especially
when the amount of the message is around the level of the limit of detection
(e.g. Fig. 2D), we evaluated
our results with EST information derived from the egg cDNA library. For this
analysis, only 324 genes that have at least one EST in our whole EST
collection derived from 12 different libraries
(Satou et al., 2002b) were
used. As shown in Table S14 (at
http://dev.biologists.org/supplemental),
of the 10 genes with mRNA localized in the posterior region of the egg [such
as posterior end mark (pem)
(Yoshida et al., 1996
)], seven
genes have one or more corresponding ESTs derived from the egg cDNA library
and the remaining three genes do not have any corresponding ESTs. The maternal
expression of these 10 genes is most reliable, because of their localized
signal. This suggests that nearly 30% of maternal genes are likely to
represent genes with rare maternal message and are not included in the EST
data. Therefore, if our in situ data are correct, a similar percentage of the
EST coverage is expected for genes with unlocalized mRNA. In fact, of 224
transcription factor genes with unlocalized maternal mRNA, 163 genes (73%)
have one or more ESTs and the remaining 61 genes (27%) do not. A similar
percentage of the presence of the corresponding ESTs indicates accuracy of the
result. Of 90 genes in the `genes not maternally expressed' class, 84 genes
(93%) do not have any corresponding ESTs and the remaining six genes (7%) have
one or two corresponding ESTs. The number of the corresponding ESTs for each
of these six genes was two at maximum and 1.3 on average, while the number of
the ESTs for each gene with maternal expression is 30 at maximum and 3.2 on
average, suggesting a possibility that these six genes are maternally
expressed at low level below the limit of detection by in situ hybridization.
The evaluation with the EST data overall supports the reliability of the in
situ result.
These two methods used in the present study have merits and demerits, respectively. The EST analysis could miss rarely expressed genes if the number of the ESTs is not large enough, while the EST analysis does not have a limit of detection if the number of the ESTs is large enough. The above comparison between results of the in situ hybridization and the EST analysis indicates our EST collection still misses some rarely expressed genes, although C. intestinalis is ranked sixth in the number of ESTs among all animals (NCBI EST database, May 7, 2004). However, in situ hybridization has a limit of detection and the result has to be evaluated visually, which means it is less objective than the EST analysis. However, in situ hybridization has the advantage of being able to test any genes accurately, even if the genes are rarely expressed. Considering the advantages and disadvantages of both methods, the inconsistentcy (3+61+6=70 genes; 22%) of both results probably corresponds to the rarely maternally expressed genes and this inconsistency may never be reconciled even in future studies. Nevertheless, the overall consistency (78%) supports the overall reliability of the in situ result. Accordingly, 52% of transcription factor genes (163+7=170 genes) are maternally expressed, 20% (3+61=64 genes) are maternally expressed at a low level (detectable by in situ hybridization), 2% (6 genes) are maternally expressed at a very low level (undetectable by in situ hybridization) and 26% (84 genes) are not maternally expressed.
Empirically, expression below the limit of detection by in situ hybridization is not so important for ascidian embryogenesis. Therefore, we discuss the maternal expression based on the results of the in situ hybridization of the 352 transcription factor genes, including 28 genes whose cDNA was isolated by PCR (these were not included in the comparison with the EST data). As summarized in Fig. 3A, 74% (259 genes) of transcription factor genes are expressed maternally, of which 3% (10 genes) are localized in the posterior region of the egg, and the remaining 26% (93 genes) of transcription factor genes were not detected maternally. In other words, a significant percentage of the Ciona transcription factor genes (74%) are expressed maternally above the limit of detection by in situ hybridization. It is thought that the dependency of development on maternally supplied information is higher in the ascidian than in other animals, because the ascidian egg shows a `mosaic' mode of early embryogenesis. Therefore, this high percentage of maternally expressed transcription factor genes may be specific for ascidian development, but this notion should be tested by comparing results from other model animals in future. Conversely, if the unveiled maternal determinants are a transcription factor like the macho-1 gene in Halocynthia, candidates are only 259 genes described here.
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Zygotically expressed transcription factor genes
Of 352 transcription factor genes examined, 199 (56%) genes are expressed
zygotically during Ciona embryogenesis
(Fig. 3C; Tables S6-S13). Of
these 199 genes, 120 genes (34%) are also expressed maternally and the
remaining 79 genes (22%) are expressed only zygotically. Of genes that are not
expressed zygotically, 14 genes (4%) are not expressed maternally. Therefore,
only 14 genes are not used during embryogenesis.
Fig. 3D shows the number of
zygotically expressed genes at each developmental stage. At the 16-cell,
32-cell, 64-cell, early gastrula, late-gastrula, neurula, early tailbud and
mid-late tailbud stage, 13, 23, 47, 58, 90, 129, 175 and 180 genes are
zygotically expressed, respectively. The number of genes expressed gradually
increases as development proceeds. This may be simply explained by a
hypothesis that the construction of a body with more complex elements requires
more transcription factor genes.
Figure 3E,F shows the number and the ratio of the expressed gene number of each of seven major transcription factor families. Each transcription factor family may have a tendency in its zygotic expression according to the developmental stage. Fox transcription factor genes show a tendency for the genes to begin to be expressed at earlier stages, while the bHLH, HD and Ets families show a tendency for initiation of their expression to occur at later stages. A lower proportion of bZip, HMG and NR family genes are zygotically expressed during Ciona embryogenesis. Therefore it is possible that some transcription factor family may be largely assigned specific roles in development, although the number of genes in each gene family is not large enough to obtain statistically meaningful conclusions. Because the number of transcription factor genes in one animal is limited, this should be further examined in other animals in future.
Genes for signaling molecules
We previously identified most of the major signaling molecule genes in the
Ciona intestinalis genome, including all ligands and major components
of the Wnt, TGFß, Fgf, Notch, Hedgehog and JAK/STAT pathways [Tables
2, S2
(http://dev.biologists.org/supplemental)]
(Satou et al., 2002c;
Satou et al., 2003b
;
Hino et al., 2003
). Embryonic
expression profiles of these genes are also described here. The initial list
includes 118 genes, of which cDNAs for 109 genes were obtained for analysis
similar to that performed for transcription factor genes
(Table 2). The analysis
revealed that 83 signaling genes are expressed maternally and six genes of
them are localized in the posterior end of the embryo (Table S5 at
http://dev.biologists.org/supplemental).
Sixty-eight genes are zygotically regulated at the transcription level (Tables
S6-S13 at
http://dev.biologists.org/supplemental).
For example, Dickkopf is not expressed maternally or at the 16-cell
stage, but is expressed in A6.1, A6.3 and B6.1 at the 32-cell stage, in A7.1,
A7.2, A7.5, B7.1 and B7.2 at the 64-cell and early gastrula stages, and in
endodermal cells and cells of the nervous system at the late gastrula stage
and thereafter (Fig. 2E).
Wnt5 is maternally expressed and localized to the posterior pole of
the embryo, as is pem (Fig.
2F). The zygotic expression of Wnt5 begins in B6.1 and
B6.2 at the 32-cell stage, and is seen subsequently in A7.4, A7.8, B7.3, B7.4,
B7.7 and B7.8 at the 64-cell stage, B7.7, B8.5, B8.7, B8.8, B8.15 and B8.16 at
the early gastrula stage, muscle and posterior epidermis at the late gastrula
and neurula stages, and parts of epidermis at the tailbud stage
(Fig. 2F).
We compared these descriptions with those of genes analyzed in the previous
studies, including Fgfs (Imai et al.,
2002c; Imai et al.,
2002d
), lefty/antivin
(Imai, 2003
),
hedgehog1 and hedgehog2
(Takatori et al., 2002
). Our
descriptions are basically same as the previous ones, indicating that the
present assay is reliable. However, an apparent discrepancy in one gene was
found. hedgehog1 was not found to be zygotically expressed in the
previous study, but in the present study the gene was found to be zygotically
expressed in the 32-cell stage embryo and in embryos from the early gastrula
stage to the mid-late tailbud stage, which was confirmed by two independent
experiments. But the expression was very weak, and therefore we speculate that
recent advances in the in situ hybridization techniques and methodology have
made this expression detectable, although the precise reason is unknown.
Possible transcription factor networks
In ascidian development, the developmental fate of almost all blastomeres
is restricted to one tissue at or before the 110-cell stage, as described
above. The present comprehensive description demonstrated that only 65
transcription factor genes and 25 signaling genes are zygotically expressed
until the early gastrula stage. Namely, the Ciona embryo requires
only 65 zygotically expressed transcription factor genes and 25 signaling
genes for the embryonic tissue specification. Therefore, we may be able to
reconstruct comprehensive transcriptional networks by analyzing this limited
number of genes in future.
Figure S1 shows transcription factor genes zygotically expressed during early embryogenesis in individual tissue lineages at each developmental stage. For example, the ascidian endoderm is determined by Lhx3, which is activated directly or indirectly by maternal ß-catenin. Lhx3 is first expressed in cells with endodermal fate at the 32-cell stage (Table S7; Fig. S1). The present analysis showed that only 12 transcription factor genes are zygotically expressed in this lineage of cells at the 16-cell stage and only 15 genes, except Lhx3 itself, are expressed at the 32-cell stage (see Fig. S1 at http://dev.biologists.org/supplemental). Therefore, analyzing genetic relationships among only 18 genes (=12+159; nine genes are expressed both at the 16 and 32-cell stage) clarifies a zygotic gene network beginning with maternal ß-catenin and leading to activation Lhx3 in the endodermal lineage for its specification.
Table S15 (see
http://dev.biologists.org/supplemental)
shows transcription factor genes whose expression overlaps that of individual
transcription factor genes and signaling genes zygotically expressed in early
embryogenesis at each developmental stage (i.e. possible direct
transcriptional regulators of each of transcription factor genes and signaling
genes are shown, on the assumption that each transcription factor protein has
the same life span as its mRNA). For example, Table S15
(http://dev.biologists.org/supplemental)
shows that 13 transcription factor genes are expressed in the blastomeres
where Brachyury is first expressed at the 64-cell stage. In fact, one
of these 13 genes, ZicL, was recently shown to be a direct activator
of Brachyury (Yagi et al.,
2004), suggesting that this type of comprehensive description will
become an important milestone for future studies of the ascidian
development.
Genes downstream of ß-catenin, FoxD and Fgf9/16/20
As a first step to reconstruct the transcriptional gene network involved in
making the ascidian endomesoderm specification, we chose three important
genes; ß-catenin, FoxD and Fgf9/16/20. As described
above, ß-catenin is essential for endoderm differentiation, and therefore
required for forming tissues such as notochord and mesenchyme, which are
induced by endodermal cells (Imai et al.,
2000). FoxD is expressed under the control of
ß-catenin and essential for specification of notochord and TLCs
(Imai et al., 2002b
;
Imai et al., 2003
).
Fgf9/16/20 is also expressed under the control of ß-catenin and
induces mesenchyme and is partially responsible for inducing notochord
(Imai et al., 2002c
).
Therefore, genes under the control of these three genes should be involved in
notochord specification. Genes under the control of ß-catenin and
FoxD but not Fgf9/16/20 should be involved in specification
of notochord or TLCs. Genes under the control of ß-catenin and
Fgf9/16/20, but not FoxD should be involved in specification
of mesenchyme or cells of the nervous system. Genes under the control of
ß-catenin but not FoxD and Fgf9/16/20 should be
involved in specification of endoderm.
We checked whether or not each of transcription factor and signaling genes that are expressed at the early gastrula stage is down- or upregulated by suppression of any of these three genes. For this, the real-time PCR method was adopted, because the quantity of each mRNA can be more precisely measured by this method than by microarray analysis when analyzing a limited number of genes. Only when at least two independent experiments indicated more than threefold difference in the amount of the gene transcript between control embryos and experimental embryos, the gene was counted as one that is affected by suppression of any of the three genes (Table S16; Fig. S2).
As summarized in Fig. 4, 26 transcription factor genes and 11 signaling genes were downregulated and one transcription factor gene was upregulated as a result of suppression of ß-catenin. Five transcription factor genes and one signaling gene were downregulated, and two transcription factor genes and one signaling gene were upregulated by suppression of FoxD; and nine transcription factor genes and one signaling gene were downregulated by suppression of Fgf9/16/20. All genes that we previously identified as downstream genes of any of these three genes by in situ hybridization were included (see below), validating this analysis.
|
Among genes downstream of ß-catenin and FoxD but not
Fgf9/16/20, which are likely to be involved in specification of
notochord and/or TLCs, ZicL, which is essential for notochord
specification via activation of Brachyury transcription, was included
(Imai et al., 2002a;
Yagi et al., 2004
). Among
these genes, Mnx is specifically expressed in the notochord lineage,
E(spl)/hairy-b is expressed in the secondary-lineage (B-line)
notochord cells and a part of nerve cord cells, and FoxB and
Fgf8/17/18 genes are expressed in cells of the TLC lineage. The
expression pattern coincides well with the expected functions of these
genes.
Genes downstream of ß-catenin and Fgf9/16/20 but not
FoxD are probably involved in specification of mesenchyme, including
TLCs and/or cells of the nervous system. Among these genes, Twist-like-1,
Orphan bZIP-4, Fli/ERG1, Jun, Fos and chordin are expressed in
mesenchymal cells. Ciona savignyi Twist-like-1 has been proven to be
essential for the specification of mesenchyme
(Imai et al., 2003).
Myelin transcription factor (MyTF) is expressed in cells of
the TLC lineage. Chordin is also expressed in notochord cells and a
part of muscle cells. DMRT1 and Msxb genes are expressed in
cells of the nervous system.
Genes downstream of ß-catenin are probably involved in other vegetal
cell specification, including specification of endoderm. Lhx3, which
is one of these genes, is known to play an essential role in differentiation
of endoderm (Satou et al.,
2001a). The gene for TTF1, which is also known to be involved in
endoderm specification, is also included in this group
(Ristoratore et al., 1999
).
The common feature of this class of genes is that all genes except nodal are
expressed in one or more vegetal cells (Tables S5-S13).
Thus, the mode of regulation summarized in
Fig. 4 and the expression
pattern summarized in Fig. S1 (see
http://dev.biologists.org/supplemental)
of each gene will strongly predict its function. Therefore the present data
will also become another important milestone for future studies of gene
function. We cannot know from this analysis whether all expression of a given
gene was affected or a part of the expression was affected. For example, it
was shown by in situ hybridization that the expression of ZicL in
A-line cells is affected by ß-catenin but the expression in B-line cells
is not (Imai et al., 2002a).
In spite of this weakness, ZicL was still detected to be suppressed
by ß-catenin morpholino in the present real-time PCR experiment,
suggesting usefulness of this analysis in constructing frameworks of gene
networks. In future studies, similar real-time PCR experiments using embryos
at different stages should be performed. Once genes are identified as
downstream of ß-catenin, FoxD and/or Fgf9/16/20, the
function of the genes should be determined and their epistatic relationships
will be determined in a similar way. Thus, these approaches will reveal
frameworks consisting of transcription factor genes and signaling genes for
the complete understanding of the molecular mechanisms behind ascidian
endomesoderm specification.
Conclusion
As discussed here, the expression profiles of Ciona intestinalis
transcription factor genes and major signaling genes have been almost
completely determined from the egg to the mid-late tailbud stage embryo. The
patterns thus revealed answer several fundamental questions about how the
transcription factor genes are used to build up the basic chordate body plan.
In the ascidian embryo, almost all blastomeres are specified and determined at
or before the 110-cell stage. The present analysis performed at the
single-cell level has shown that 65 transcription factor genes are zygotically
expressed at or before this stage. In other words, the ascidian embryo
requires only 65 transcription factor genes to be zygotically expressed for
specification of larval tissues. This number is smaller than expected. Thus,
further analysis of this very limited number of transcription factor genes
will allow elucidation of the complete transcriptional networks that are
essential for the tissue specifications at the single-cell level.
Possible regulatory relationships among the transcription factor genes can be deduced from the present study, exemplified by Table S15, although we have not yet reconstructed any gene networks. These possible relationships can be actually confirmed by mid-to-high throughput analysis such as real-time PCRs adopted in the present study.
To obtain a real understanding of the molecular mechanisms behind animal development, comprehensiveness is essential. Quantitative comprehensiveness can be easily attained using modern methods such as microarrays and real-time PCR. However, because animal development is controlled spatially and temporally, qualitative comprehensiveness is also required. To obtain these data is tedious, and therefore to start studies with a subset of genes would be a good alternative. We present here such comprehensive qualitative data about transcription factor genes and signaling genes in several major signaling pathways. These data will provide an important scaffold for achieving complete understanding of ascidian embryogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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