1 Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku,
Kyoto 606-8502, Japan
2 CREST, Japan Science and Technology Agency, Tokyo, Japan
Author for correspondence (e-mail:
satoh{at}ascidian.zool.kyoto-u.ac.jp)
Accepted 2 September 2003
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SUMMARY |
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Key words: Ciona intestinalis, Reverse genetics, Novel genes, Morpholino oligonucleotide, Translational suppression
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Introduction |
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Urochordate ascidians are marine invertebrate chordates that have a common
ancestor shared by the cephalochordate amphioxus and vertebrates. The
organization of ascidian tadpole larvae shows basic features of the chordate
body plan (Satoh, 1994;
Corbo et al., 2001
;
Satoh, 2003
). The ascidian
genome has been proposed to contain a basic set of chordate-type genes
corresponding to those present before the large-scale gene duplications
occurred in the lineage leading to vertebrates
(Holland et al., 1994
), and
thus ascidian genes have less functional redundancy than vertebrate genes.
Recently, the draft genome sequence of the most-studied ascidian, Ciona
intestinalis, has been reported (Dehal
et al., 2002
). Its 159 Mbp genome (17 times smaller than that of
humans) contains 15,852 protein-coding genes, similar to the number in other
invertebrates but only half that found in vertebrates. Vertebrate gene
families are typically found in simplified form in the Ciona
intestinalis genome, supporting the idea that ascidians contain the basic
ancestral complement of genes involved in cell signaling and development
(Dehal et al., 2002
;
Satou et al., 2003a
;
Wada et al., 2003
;
Yagi et al., 2003
). In
vertebrates, the function of a gene is often obscured by functional redundancy
of the related genes. Therefore, Ciona embryos may provide an
appropriate experimental system for exploring the functions of genes
(Satoh et al., 2003
).
The Ciona intestinalis cDNA project consortium has conducted
comprehensive studies of gene expression profiles in fertilized eggs
(Nishikata et al., 2001),
cleavage stage embryos (Fujiwara et al.,
2002
), tailbud embryos (Satou
et al., 2001b
), larvae
(Kusakabe et al., 2002
) and
young adults (Ogasawara et al.,
2002
). A total of more than 480,000 ESTs have been sequenced, and
the spatial expression profiles of 5000 randomly selected genes have been
determined (Satou et al.,
2002a
; Satoh et al.,
2003
). The genes have been classified into three major classes
according to the function of the proteins they encode
(Lee et al., 1999
). Class A
includes genes associated with functions in many cell types, class B contains
genes associated with cell-cell communication and class C contains genes that
function as transcription regulatory proteins. Besides genes that fall into
these three classes, there are many genes for which not enough information is
available to determine their biological function. These genes have been
categorized into two classes, class DI and class DII. Class DI includes
sequences that match ESTs or reported proteins with unknown function (mostly
from humans and mice), and class DII consists of genes with no significant
sequence similarity to known genes. Ciona intestinalis cDNA analysis
suggests that at least 2500 genes are classified into class DI, and that
nearly one quarter of them show specific spatiotemporal expression patterns.
Most DI-class genes are assigned counterparts of vertebrate genes with unknown
function. Therefore, a comprehensive functional analysis of Ciona
intestinalis DI-class genes would provide significant information
relevant to vertebrate genes with unknown function. We therefore decided to
systematically investigate the loss-of-function phenotypes of DI-class genes,
starting with 200 genes in the present pilot screen.
In addition to the small size of their genome and the small number of
genes, ascidians provide a simple experimental system for investigating the
molecular mechanisms underlying cell-fate specification during chordate
development (Satoh, 1994;
Satoh, 2001
). The Ciona
intestinalis fertilized egg develops within 18 hours into a tadpole
larva, through invariant bilateral cleavage, gastrulation, neurulation and
tailbud formation. The lineages of embryonic cells are invariant among
individuals and have been well documented
(Conklin, 1905
;
Nishida, 1987
). The tadpole
larva consists of
2600 cells that form distinct types of tissues and/or
organs, including epidermis that covers the entire surface; a central nervous
system with two sensory pigment cells; endoderm and mesenchyme in the trunk
and notochord; dorsal nerve cord; ventral endodermal strand; and muscle in the
tail (Fig. 1A). The Ciona
intestinalis cDNA project has provided us with genes expressed in a
tissue-specific manner for almost all types of tissue
(Satou et al., 2001b
;
Kusakabe et al., 2002
). These
genes can be used as tissue-specific markers. Furthermore, over 5600
full-length sequences of cDNAs have already been determined
(Satoh et al., 2003
). These
circumstances make Ciona intestinalis embryos a suitable system for a
reverse genetic approach for identifying novel genes with developmental
function.
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In the present study, we conducted an extensive analysis of genes discovered in the Ciona intestinalis cDNA project. To identify genes with developmental function, translation of a total of 200 genes expressed in embryos was suppressed using specific morpholinos. Most of these 200 genes were classified into class DI, genes whose vertebrate counterparts have unknown function. The suppression of the translation of any of 40 of these genes (20% of the genes examined) caused specific embryonic defects. These results demonstrate that the Ciona embryo is a powerful tool for identifying developmental genes essential for formation of the chordate body plan. Furthermore, the present study might represent the first example of a screen using morpholinos as a reverse genetic tool to identify genes required for chordate development.
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Materials and methods |
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Characterization of genes for the screen
For the 200 genes examined in the present study, clones of the
corresponding cDNA that encoded the full length of the putative protein were
isolated and sequenced as described previously
(Satou et al., 2002b). In all
of these cDNA clones, there are in-frame stop codon(s) upstream of the
putative initiation codon, which supports the validity of the prediction of
the initiation codon. Motifs in the putative proteins were searched with SMART
(http://smart.emblheidelberg.de/).
Homology searches were performed against the DDBJ nucleotide database with
BlastX, and gene sequences with P values less than 10-15
were selected as homologs. Temporal expression profiles of genes were
determined based on the results of the EST analyses
(Satou et al., 2002a
). A list
of the 200 genes including gene collection IDs, clone IDs (used as the name of
each gene here), Accession Numbers of cDNA sequences and nucleotide sequences
of the corresponding morpholino are provided in Table S1 at
http://dev.biologists.org/supplemental.
EST information and the cDNA sequences of the 200 genes are available in our
web site
(http://ghost.zool.kyoto-u.ac.jp/indexr1.html).
Design of morpholinos and microinjection
Morpholinos were made to order (Gene Tools, LLC). For most of the 200
genes, a complementary morpholino was designed to target a sequence containing
the putative initiation codon. For the other genes, a morpholino was designed
to target a sequence of the 5' untranslated region (Table S1 at
http://dev.biologists.org/supplemental).
Microinjection was carried out as described previously
(Satou et al., 2001a). The
amount of morpholino injected was 10 fmoles, an amount chosen based on the
results of previous reports (Satou et al.,
2001a
) and our preliminary experiments. Ten fmoles of morpholinos
dissolved in distilled water were injected into fertilized eggs and the
effects on morphology were determined using stereomicroscopy about 18 hours
later at the tadpole larva stage. In cases of the use of a second form of
citb018a19 morpholino, 30 fmoles were injected into fertilized eggs. As
controls, 10 or 30 fmoles of a morpholino against lacZ were injected,
but these had no effect on embryogenesis, provided excess amounts were not
injected.
Two researchers simultaneously, but independently, injected morpholinos into 25 or more fertilized eggs. When they obtained the same result, it was scored. When the results of the two researchers were inconsistent, a third researcher performed another injection to reach a final conclusion.
Rescue experiments with synthetic mRNA
For rescue experiments, the protein-coding region of Ci-Bra,
cieg003h01 or citb018a19 that lacked a target sequence for morpholino was
amplified by PCR and cloned into pBluescriptRN3 vector. Capped mRNA was
synthesized in vitro using a Megascript T3 kit (Ambion) together with the cap
analog 7mGpppG. Because the synthesized mRNA lacks the target sequence for the
corresponding morpholino, translation from it is not inhibited by the
morpholino. The fertilized eggs were injected with 5 pg (Ci-Bra) or
25 pg (cieg003h01 and citb018a19) of the synthesized mRNA together with 10
fmoles of the corresponding morpholino, allowed to develop up to the tailbud
embryo stage, and examined for marker gene expression or morphology.
Whole-mount in situ hybridization and histochemistry
Whole-mount in situ hybridization was carried out as described previously
(Satou et al., 2001b). The
probes used were Ci-Epi1 for epidermal cell differentiation
(Chiba et al., 1998
),
Ci-ETR for nervous system development
(Satou et al., 2001b
),
Ci-talin for notochord differentiation
(Satou et al., 2001b
;
Sasakura et al., 2003
) and
ciad005j06 for mesenchyme cell differentiation
(Satou et al., 2001b
). Probes
for Ci-arr (Nakagawa et al.,
2002
), Ci-opsin1
(Kusakabe et al., 2001
),
Ci-opsin3 (Nakashima et al.,
2003
) and Ci-G
i1a
(Yoshida et al., 2002
) were
also used for experiments with the citb018a19 gene.
Histochemistry for endoderm alkaline phosphatase (ALP) and muscle
acetylcholine esterase (AchE) was performed as described
(Imai et al., 2000).
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Results |
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Translational suppression of each gene was achieved by microinjection of a
corresponding morpholino into fertilized eggs, and the resultant embryos were
examined for any defect in larval morphology to identify genes required for
normal development. When morpholino against lacZ mRNA was injected as
a negative control, all of the injected embryos developed normally (data not
shown). However, injection of morpholino complementary to Ci-Bra, an
essential regulator of notochord development in Ciona embryos
(Corbo et al., 1997;
Takahashi et al., 1999
), at
the same dose led to a shortened tail phenotype in almost all injected embryos
(Fig. 1B). Expression of
Ci-talin, a notochord marker (Fig.
1C) was lost in the morpholino-injected embryos
(Fig. 1D). These findings are
consistent with the proposed role of Ci-Bra in notochord formation.
The specificity of the action of Ci-Bra morpholino was assessed by a
rescue experiment with synthetic Ci-Bra mRNA. Co-injection of
Ci-Bra morpholino and Ci-Bra mRNA abrogated the loss of
Ci-talin expression caused by injection of Ci-Bra morpholino
(Fig. 1E).
A gene was considered to be positive when half or more of the specimens
within the group of injected embryos exhibited developmental deficiency. Out
of the 200 genes examined, 40 genes were judged to be positive by this
criterion (Table 1; information
on the 40 positive genes is summarized in
Table 2). The remaining 160
genes were judged to be tentatively negative and excluded from the subsequent
analysis. It has been reported that, in zebrafish embryos, the penetrance of
the effects of morpholinos is variable and much lower than 100% in many cases
(Heasman, 2002). Similarly, in
our experimental system, the efficiency of morpholinos judged to be positive
was variable depending on the morpholino. We estimated that the efficiency
(the rate of embryos showing a developmental defect in the injected embryos)
was in the range 60-100%, depending on the morpholino, although the reason for
this variation is unknown. In Table
2, morpholinos with efficiency of 80% or higher were considered to
have strong penetrance, while those with lower efficiency were considered to
have weak penetrance.
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Genes associated with the `disorganized body plan' phenotypic
class
To provide an overview of the outcome of the present screen and to
illustrate the morphological deficiencies of embryos, several interesting
examples are described in the following two sections. Fourteen genes were
associated with the `disorganized body plan' phenotypic class
(Table 1). They consisted of 12
DI-class genes, including eight genes for Zn-finger proteins, one DII-class
gene and one C-class gene that is a Ciona intestinalis homologue of
Pbx (Table 2). Several
intriguing examples in this phenotypic class are described.
cieg015c02 encodes a protein that contains C2H2 type Zn-finger motifs and shows sequence similarity to the human hypothetical protein XP 030938 (Table 2). ESTs for this gene were detected in fertilized eggs and cleaving embryos. Eggs injected with its morpholino developed into rough-surfaced aggregates of cells (Fig. 2A). They were not enclosed by a smooth-surfaced epidermis layer. The cells that comprised them were loosely connected, so that the aggregates were very fragile. In spite of such extreme abnormality, the expression of markers for some endodermal and mesodermal tissues was detected in them, suggesting that they did not simply expire. Rather, it seems likely that a mechanism involved in cell adhesion or epidermis formation is affected in the cieg015c02 knockdown embryos.
cieg040a13 encodes a protein that has a Zpr1 motif
(Galcheva-Gargova et al.,
1998) and is similar to the human Zn-finger protein 259
(Table 2). ESTs for this gene
were scored in fertilized eggs, cleaving embryos and young adults. Eggs
injected with morpholino complementary to this gene developed into deformed
embryos (Fig. 2B). The size of
the cells constituting these embryos was significantly larger than that of any
cells comprising normal embryos, suggesting that the process of cell division
was affected in these embryos.
Two distinct genes, cicl045c22 and cieg003h01, gave similar, remarkable phenotypes. cicl045c22 encodes a protein that contains C2H2 type Zn-finger motifs and is similar to the human hypothetical protein XP_ 041139 (Table 2). ESTs for this gene were detected at all the developmental stages examined. Eggs injected with cicl045c22 morpholino or cieg003h01 morpholino developed into flattened larvae with similar appearance (Fig. 2C,D). Here, the results of suppression of cieg003h01 function are described in detail.
cieg003h01 encodes a protein that has multiple putative transmembrane
motifs and is similar to the human hypothetical protein FLJ12377
(Table 2) and its homologs are
present in the genomes of mice, flies, worms, plants and yeasts
(Table 2). ESTs for this gene
were detected at the stages of fertilized egg, cleaving embryo and larva, and
its maternal message is distributed evenly in the egg cytoplasm (data not
shown). Eggs injected with cieg003h01 morpholino developed into flattened
larvae with trunk-like and tail-like regions
(Fig. 2D,
Fig. 4D), although the cleavage
pattern became atypical as early as the eight-cell stage. The trunk-like
region appeared to comprise a layer of epidermal cells with no visible
development of the endoderm and nervous system
(Fig. 2D). Whole-mount in situ
hybridization revealed the expression of Ci-Epi1, an epidermal cell
differentiation marker (Fig.
4A,B), and histochemistry demonstrated the production of muscle
AchE (Fig. 4C,D). In ascidian
embryos, the differentiation of epidermis and muscle takes place autonomously
dependent on maternally provided egg cytoplasmic information (e.g.
Nishida, 2002). This suggests
that the genetic cascades leading to the differentiation of epidermis and
muscle are independent of cieg003h01. However, in the trunk-like region of
experimental embryos, neither the endoderm assessed by histochemical detection
of ALP (Fig. 4K,L) nor the
nervous system assessed by Ci-ETR expression
(Fig. 4G,H) differentiated. In
the tail-like region, the differentiation of notochord was inhibited
(Fig. 4I,J) and that of
mesenchyme was partially suppressed (Fig.
4E,F). The differentiation of notochord and mesenchyme is
dependent on the endoderm in Ciona embryos
(Imai et al., 2000
;
Imai et al., 2002a
). The
specificity of the action of cieg003h01 morpholino was assessed by a rescue
experiment with synthetic cieg003h01 mRNA. Co-injection of the morpholino and
the mRNA abrogated the loss of ALP expression caused by the injection of
cieg003h01 morpholino (Fig.
4N). In some cases, the co-injection resulted in the formation of
larvae with a deformed but distinct tail region
(Fig. 4M).
|
cilv008a08 is the single Ciona intestinalis counterpart of
vertebrate Pbx genes (Wada et al.,
2003) (Table 2).
ESTs for this gene were detected in all the developmental stages examined
(Table 2). Eggs injected with
cilv008a08 morpholino developed into ball-shaped larvae
(Fig. 2E). Pbx proteins are
TALE class homeodomain transcription factors that act as cofactors for Hox
class homeodomain transcription factors and play multiple roles during
development according to their partner proteins
(Mann and Chan, 1996
). The
severe defects observed in the cilv008a08 morpholino-injected embryos suggest
that cilv008a08 may play a similar crucial role.
In addition to the genes described above, nine genes gave the `disorganized body plan' phenotype. Although they are not described in detail here, embryos injected with morpholino against each of them are shown in Fig. 2F-N.
Genes associated with the `abnormal tail' phenotypic class
Of the 40 positive genes, 26 genes belonged to the `abnormal tail'
phenotypic class. They consisted of 15 DI-class genes, six DII-class genes,
two A-class genes, two B-class genes and one C-class gene, and included six
genes encoding Zn-finger proteins (Table
2).
cieg022g05 encodes a protein that has four SANT motifs
(Aasland et al., 1996) and
shows sequence similarity to the human SNAP190 protein, the function of which
is unknown (Table 2). ESTs for
this gene were detected in all the developmental stages examined. Eggs
injected with cieg022g05 morpholino developed into larvae that had bent tails
and lacked the sensory pigment cells (Fig.
3A). Remarkably similar phenotypes were obtained when eggs were
injected with morpholino that targets cieg015b23, which encodes a protein
similar to the human KIAA1629 protein (Fig.
3B), or cicl057g13, which encodes a protein similar to the human
hypothetical protein FLJ12890 (Fig.
3C), although the average length of the tail varied depending on
the morpholino. On the other hand, injection of a morpholino designed against
cilv006d23, which encodes a protein with a BTB motif
(Zollman et al., 1994
) that is
similar to the human hypothetical protein MGC2628
(Table 2), also led to larvae
with a bent tail; the larvae developed the sensory pigment cells but lacked
the palps (Fig. 3D).
cicl002e04 encodes a protein that belongs to the Zic family of C2H2-type
zinc-finger proteins (Table 2).
ESTs for this gene were detected in embryos at the cleavage, gastrula/neurula
and tailbud stages. Zic family genes play crucial roles in development in a
wide range of animals (Nagai et al.,
1997). Eggs injected with cicl002e04 morpholino developed into
larvae that had short tails and had palps but no sensory pigment cells
(Fig. 3E). Recently, Zic family
genes were isolated from Ciona savignyi (Cs-ZicL)
(Imai et al., 2002b
) and
Halocynthia roretzi (HrzicN)
(Wada and Saiga, 2002
), and
translational suppression experiments using a specific morpholino showed that
larvae injected with Cs-ZicL or HrzicN morpholino exhibited
defects in notochord and neural tube development. A molecular phylogenetic
analysis of ascidian Zic family genes suggests that cicl002e04 is one of the
multiple Ciona intestinalis counterparts of Cs-ZicL and
HrzicN (Yamada et al.,
2003
). Consistent with this, the knockdown phenotypes reported for
Cs-ZicL and HrzicN were similar not only to each other but
also to those of animals injected with cicl002e04 morpholino.
Here, we describe in detail the results obtained with citb018a09.
citb018a09 encodes a protein that has a Ring finger motif and a WWE domain,
which shows sequence similarity to the human hypothetical protein
DKEZp434O1427 with unknown function (Table
2). ESTs for this gene were detected in all the embryological
stages but not in the adult (Table
2). Eggs injected with citb018a09 morpholino (a form that was
produced against 25 nucleotides, including the first ATG codon) developed into
larvae that had lacked the sensory pigment cells
(Fig. 5B). Whole-mount in situ
hybridization and histochemistry revealed the occurrence in the
morpholino-injected embryos of differentiation markers for epidermis
(Ci-Epi1), endoderm (ALP), muscle (AchE), notochord
(Citalin) and mesenchyme (ciad005j06) (data not shown). Expression of
pan-neural marker (Ci-ETR) indicated that citb018a09 is not required
for neural tube formation (Fig.
5D,E). Because citb018a09 is likely to be involved in the
formation of the sensory organs, we also examined the effects of its
functional suppression on the photosensory organ. The expression of four genes
involved in the function of the photosensory organ, Ci-opsin3
(Fig. 5F,G), Ci-arr
(Fig. 5H,I), Ci-opsin1
(data not shown) and Ci-Gi1a (data not shown)
appeared in citb018a09-morpholino-injected embryos as in normal tailbud
embryos. Co-injection of citb018a09 morpholino and citb018a09 mRNA failed to
rescue the formation of pigment cells. However, a second form of citb018a09
morpholino which was produced against the 5'UTR also gave the same
results as the first morpholino. These results suggest that this gene is not
likely to be involved in the formation or function of the photosensory organ,
but rather in pigment cell formation.
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Discussion |
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Evaluation of the present screen for novel developmental genes in
Ciona intestinalis embryos
It has been shown that morpholinos often exhibit nonspecific side effects
on embryogenesis in zebrafish (Heasman,
2002). One of the conceivable reasons for this is unexpected
targeting of other genes by the morpholinos. However, this is not likely to
have been the case in the present screen, because we confirmed that, for each
morpholino that induced any phenotype studied here, there was no gene that had
a complementary sequence around or upstream of the initiation codon in the
Ciona intestinalis genome other than the original target gene (data
not shown). Another possible reason for this type of side-effect of
morpholinos is nonspecific effects of morpholinos or contaminants. As
mentioned above, however, the alteration of the morphology induced by a given
morpholino seemed to be specific and distinct from that induced in embryos
injected with Ci-Bra morpholino. Further support for the specificity
of the action of morpholinos was obtained by rescue experiments. Therefore, it
is likely that the phenotypes obtained in the present study were caused by the
specific actions of the morpholinos rather than nonspecific side effects of
morpholinos. The successful identification here of two genes that are expected
to be involved in development, a Zic family gene (cicl002e04) and a homologue
of Pbx (cilv008a08), also suggest that the present screen was effective.
In the present simple screen of visible features, morpholino-injected embryos were judged to be positive or tentatively negative based on the observation of the morphology of the resultant larvae. As a result, 160 genes (out of the 200 genes examined) were judged to be tentatively negative. However, the present method may overlook some genes with developmental functions. If we examine the effects of suppressing gene activity with more sophisticated techniques and specific probes that can detect changes at the cellular and molecular level, we may be able to more efficiently identify genes with developmental functions using the present methodology.
Comparison with other genetic screens
Large-scale screens for developmental genes using a reverse genetic
approach have been conducted in the nematode C. elegans. A systematic
analysis of C. elegans chromosome I by RNAi resulted in the
functional identification of 13.9% of the genes analyzed
(Fraser et al., 2000), whereas
a similar analysis of cell division-related genes on chromosome III showed
that
6% of genes analyzed were necessary for cell division
(Gönczy et al., 2000
).
More recently, a systematic functional analysis of most of the C.
elegans genes encoded in the genome using RNAi demonstrated that 10.3% of
the examined genes showed specific phenotypes
(Kamath et al., 2003
). In
zebrafish, it has been estimated from chemical mutagenesis screens that
3-5% of the genes encoded in the genome can be mutated to yield some
developmental defects that can be identified by a visual screen of embryos
(Haffter et al., 1996
).
Compared with the findings of those screens, the efficiency of the present
Ciona intestinalis morpholino-mediated experimental system, i.e. 20%
of genes analyzed, seems appreciably higher. However, an important difference
between the C. elegans studies and the present Ciona study
should be noted. In the above-mentioned studies, a non-biased pool of genes
was screened, while in the present study the genes to be tested were selected
based on the features of their expression pattern and/or structure before the
application of the screen. This probably accounts for the difference in the
efficiency between the two screens.
In the past several years, chemical mutagenesis screening was performed in
Ciona savignyi (Moody et al.,
1999; Nakatani et al.,
1999
) and Ciona intestinalis
(Sordino et al., 2000
).
Interestingly, the spectrum of phenotypes found in those studies seems similar
to that observed in the present screen. For example, Moody et al.
(Moody et al., 1999
) reported
that the most common phenotype they obtained was `rounded', which seems to be
classified into `short tail' according to our categorization. The other
phenotypes they described were `short tail', `kinked axis (similar to `bent
tail' in the present study)', `no pigment cells' and `early arrest (probably
categorized into `disorganized body plan')', all of which are similar to
certain of the phenotypes found in the present study, although embryos with
the last phenotype show diverse features depending on the morpholino, as
discussed above, and thus are not necessarily functionally alike. The
similarities of the phenotypes obtained by the chemical mutagenesis screen and
our screen may indicate that defects in distinct developmental steps converge
to produce several types of abnormal embryos with particular morphological
features. Another notable, but probably rare, possibility is that homologous
genes are responsible for the similar phenotypes found in the various classes
of abnormal manipulated animals. In this respect, the genes identified in the
present study represent candidate genes for the mutants obtained in the
chemical mutagenesis. Further analysis is required to test these
possibilities.
Ciona intestinalis embryos provide a powerful tool to
identify novel developmental genes
Analysis of the draft genome sequence and the ESTs of Ciona
intestinalis suggests that, among a total of 15,852 genes, 10,000
genes are common to worms, flies and humans, 2600 genes are probably
chordate-specific, and another 3400 have presently been found only in
Ciona intestinalis (Dehal et al.,
2002
). As shown in Table
2, most of the DI-class genes identified in the present screen
have vertebrate counterparts with unknown function. Therefore, determining the
function of Ciona intestinalis DI-class genes may facilitate our
understanding of the function of their vertebrate counterparts. There are at
least 2500 DI-class genes in Ciona intestinalis
(Satou et al., 2002a
). A
simple estimate from the results of the present study is that about one-fifth
of DI-class genes that are expressed during embryogenesis may be shown to have
a developmental function by a morpholino-based approach. It has been shown
that most of the genes homologous between ascidians and vertebrates have a
conserved function during development
(Corbo et al., 2001
;
Satoh, 2001
). Therefore,
developmental genes suggested to have a pivotal function based on Ciona
intestinalis studies should be analyzed in Xenopus or zebrafish
embryos, or should be targeted in mouse embryos.
The cDNA project has characterized transcripts for more than three-quarters
of Ciona intestinalis genes
(Satou et al., 2002b). In
addition, more than 5600 full-length sequences of cDNAs have already been
determined. Together with the draft genome sequence, such information should
help us to design effective morpholinos. For example, in the case of a gene
that gives rise to multiple transcripts by alternative splicing, it is
possible for us to design morpholinos more carefully by taking this point into
account. The cDNA database also provides a rough estimate of the temporal
expression pattern, i.e. the count of the ESTs at each developmental stage
(Satou et al., 2003b
).
Furthermore, the spatial expression profiles of 5000 randomly selected genes
have been determined by whole-mount in situ hybridization
(Satou et al., 2002a
). These
expression profiles may help us to interpret the phenotypes of
morpholino-injected embryos. In conclusion, Ciona intestinalis
embryos provide a powerful experimental system for identifying genes with
novel developmental function required for the formation of the chordate body
plan (Satoh et al., 2003
).
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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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