1 Department of Zoology, Graduate School of Science, Kyoto University, Sakyo,
Kyoto 606-8502, Japan
2 CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 333-0012,
Japan
Author for correspondence (e-mail:
yutaka{at}ascidian.zool.kyoto-u.ac.jp).
Accepted 17 February 2004
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SUMMARY |
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Key words: Chordate, Ciona savignyi, Mesp, Heart development
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Introduction |
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Recent studies have identified genes and molecules responsible for
specification of endomesodermal cells of the ascidian embryo. Muscle cells are
specified primarily by maternal transcripts of the macho-1 gene,
which encodes a Zic-like zinc finger protein
(Nishida and Sawada, 2001;
Satou et al., 2002a
). Macho-1
is also involved in the antero-posterior patterning of Halocynthia
roretzi embryos (Kobayashi et al.,
2003
). Endodermal cells are specified primarily by maternally
provided ß-catenin (Imai et al.,
2000
), and Lhx3, which is expressed in the endodermal
lineage under the control of ß-catenin, is essential for the
differentiation of endodermal cells (Satou
et al., 2001a
). A key gene for notochord differentiation is
Brachyury (Yasuo and Satoh,
1998
; Corbo et al.,
1997
). In Ciona embryos, Fgf9/16/20
(Imai et al., 2002a
),
ZicL (Imai et al.,
2002c
; Yagi et al.,
2004
) and FoxD (Imai
et al., 2002b
) act in the upstream genetic cascade leading to
Ci-Bra expression in notochord cells. Mesenchyme cells, including
TLCs, are specified by cellular interaction, and Fgf9/16/20 plays a pivotal
role in this interaction (Imai et al.,
2002a
) and activates Twist-like1, a key gene for the
differentiation of mesenchyme cells and TLCs
(Imai et al., 2003
). However,
no genes involved in the specification of TVCs have been identified yet.
Therefore, identification and characterization of genes responsible for TVC
specification will be required for a complete understanding of the molecular
mechanisms of endomesoderm specification in the ascidian embryo.
In Halocynthia, TVCs give rise to heart, latitudinal mantle and
atrial siphon muscle in the adult (Hirano
and Nishida, 1997). The ascidian heart first appears after
metamorphosis as a tube with a single layered myoepithelium that is continuous
to a single layered pericardial wall
(Ichikawa and Hoshino, 1967
;
Satoh, 1994
;
Davidson and Levine, 2003
). The
ascidian has an open blood-vascular system, and its blood flow is regularly
reversed. Despite its structural primitiveness, the ascidian heart undergoes
morphogenesis in a similar manner to the vertebrate heart
(Davidson and Levine, 2003
). As
shown in Fig. 1A-D, in
ascidians a pair of B7.5 cells of the bilaterally symmetrical 110-cell embryo
gives rise to TVCs and a pair of anterior muscle cells in the larva. The TVCs
differentiate on both sides of the trunk of the tailbud embryo, and after
hatching they migrate and fuse along the ventral midline of the larva. After
metamorphosis, the majority of these cells are thought to differentiate to
form the heart.
|
The draft genome sequence of Ciona intestinalis suggests that the
ascidian has a much simpler genome than those of vertebrates
(Dehal et al., 2002), due
primarily to carrying fewer paralogous genes and partly to the compactness of
its intergenic regions (e.g. Satou et al.,
2003
; Wada et al.,
2003
). This simplicity greatly facilitates studies of genetic
networks. In the present study, we tried to identify genes involved in
ascidian heart formation, focusing especially on the mechanism by which early
heart progenitor cells are specified, a process that is rather difficult to
study in higher vertebrates.
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Materials and methods |
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Isolation of cDNAs and sequence determination
Ciona intestinalis cDNA clones were obtained from a `Ciona
intestinalis gene collection' (Satou
et al., 2002b). Their C. savignyi counterparts were first
searched for in genome sequences that have been produced by whole-genome
shotgun sequencing and are deposited in the Trace archive of NCBI. Based on
the genomic sequences, we amplified cDNAs from gastrula and tailbud cDNA
libraries by PCR.
Nucleotide sequences of both strands were determined using a Big-Dye Terminator Cycle Sequencing Ready Reaction kit and an ABI PRISM 377 DNA sequencer (Perkin Elmer, Norwalk, CT, USA).
Whole-mount in-situ hybridization
To determine mRNA distributions in eggs and embryos, RNA probes were
prepared using a DIG RNA labeling Kit (Roche). Whole-mount in-situ
hybridization was performed using digoxigenin-labeled antisense probes as
described previously (Satou and Satoh,
1997).
Microinjection of morpholino oligos and DiI
In the present study, we used 25-mer morpholino oligos (hereafter referred
to as `morpholinos'; Gene Tools, LLC). The sequences of the morpholinos
against Cs-Mesp were as follows: Cs-Mesp-MO1,
5'-CATGAATACGTTTCCAGGTAAAAAT-3'; and Cs-Mesp-MO2,
5'-AGATTTAAGCAAATATCGTTGCCGA-3'. The morpholinos against
ß-catenin and Cs-macho1 are described in previous
reports and their specificities have been demonstrated
(Satou et al., 2001b;
Satou et al., 2002a
).
After insemination, fertilized eggs were microinjected with 15 pmole of
morpholinos and/or synthetic capped mRNAs in 30 pl of solution using a
micromanipulator (Narishige Scientific Instrument Lab, Tokyo) as described
(Imai et al., 2000). Injected
eggs were reared at about 18°C in MFSW containing 50 µg/ml streptomycin
sulfate.
DiI (CellTracker CM-DiI, Molecular Probes) was dissolved in soybean oil at the concentration of 1 mg/ml. We injected the DiI-solution into the B6.3 blastomere of the 32-cell embryo with intact chorion to trace its lineage after metamorphosis and into the B7.5 blastomere of the 110-cell dechorionated embryo to trace its lineage to the larva. DiI-labeled embryos and juveniles were observed using fluorescent microscopy.
Detection of differentiation markers
The following cell-specific markers were used to assess the differentiation
of embryonic cells: a larval muscle-specific actin gene (Cs-MA1)
(Chiba et al., 1998), an
epidermis-specific gene (Cs-Epi1)
(Chiba et al., 1998
), a
notochord-specific gene (Cs-fibrinogen-like or Cs-fibrn)
(Imai et al., 2002a
), a
mesenchyme-specific gene (Cs-Mech1)
(Imai et al., 2002a
) and a
pan-neural marker gene (Cs-ETR)
(Imai et al., 2002a
). The
marker genes were detected by whole-mount in-situ hybridization. The
differentiation of endoderm cells in experimental embryos was monitored by the
histochemical detection of alkaline phosphatase as previously described
(Whittaker and Meedel,
1989
).
Quantitative RT-PCR
In total 25 embryos were lysed in 200 µl of GTC solution (4 M
guanidinium thiocyanate, 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 2% sarkosyl, 1%
ß-mercaptoethanol), and total RNA was prepared. The RNA was then used for
cDNA synthesis with oligo(dT) primers as described by Imai
(2003). Real-time RT-PCR was
performed using SYBR Green PCR Master Mix and an ABI prism 7000 (Applied
Biosystem). One-embryo-equivalent of cDNA was used for each real-time RT-PCR.
The cycling conditions were 15 seconds at 95°C and 1 minute at 60°C
according to the supplier's protocol. The experiment was repeated twice with
different batches of embryos. Relative expression values were calculated by
comparison with the level of expression in uninjected control embryos. Control
samples lacking reverse transcriptase in the cDNA synthesis reaction failed to
give specific products in all cases. Dissociation curves were used to confirm
that single specific PCR products were amplified.
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Results |
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Next, to confirm that Ciona TVCs give rise to adult heart, another
DiI-labeling experiment was performed. Because it is difficult to inject DiI
into the B7.5 blastomere of the 110-cell embryo with intact chorion, which is
required for metamorphosis, we injected DiI into B6.3, which is a parental
blastomere of B7.5 and B7.6, of the 32-cell embryo with chorion. The injected
embryos were incubated until they become juveniles with two gill-slits. As
shown in Fig. 1H, the heart of
the resultant juvenile was labeled with DiI, as well as several cells along
the ventral side of the stomach and degenerated larval tail muscle. Because
one of the B6.3 daughter cells, B7.6, was shown to become a germ cell, and the
primordial germ cells are aligned toward the ventral side of the stomach
(Takamura et al., 2002), the
labeled cells along the ventral side of the stomach are probably the
primordial germ cells derived from B7.6. Therefore, the juvenile heart is
highly likely to be derived from embryonic B7.5. In Halocynthia, B7.5
gives rise to latitudinal mantle muscle and atrial muscle in addition to the
heart in juveniles. However, we could not observe the labeling of these
muscles with DiI.
Identification of Cs-Mesp and its embryonic expression
In a comprehensive in-situ hybridization study of C. intestinalis
genes (Satou et al., 2002c),
we found that a sole ortholog of the vertebrate Mesp genes is
expressed specifically and transiently in heart progenitor cells at the
beginning of and during gastrulation. Because embryos of a closely related
species, C. savignyi, are more amenable to embryological
manipulations, we used C. savignyi embryos in the following studies.
First, we obtained cDNA spanning the entire coding sequence of the C.
savignyi Mesp gene (Cs-Mesp) by 5' and 3' RACE
reactions (DDBJ/EMBL/GENBANK accession number AB125640). The deduced amino
acid sequence indicated the presence of a basic helix-loop-helix (bHLH) region
that is highly conserved among Mesp family members, including mouse Mesp1,
Mesp2 and pMesogenin1. A phylogenetic tree constructed by a neighbor-joining
method revealed that the ascidian Mesp is orthologous to all known Mesp family
proteins (Fig. 2), suggesting
that the last common ancestor of ascidians and vertebrates had one gene in
this family.
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In addition, the C. intestinalis genome contains one more
HAND-like gene. Its C. savignyi ortholog was analyzed in
detail and was named NoTrlc (`no trunk lateral cells') after its
function. As was shown previously (Imai et
al., 2003), this gene is also expressed in TVCs, as well as in the
brain and in the tip of the tail at the tailbud stage
(Fig. 4C).
Furthermore, two cDNA clones for C. savignyi genes, which are
known to be expressed in the TVCs of C. intestinalis embryos
(Satou et al., 2001c), were
also isolated as molecular markers for TVCs. One gene (known by an ID number,
00152; DDBJ/EMBL/GENBANK accession number AB125643) encodes a protein with no
significant similarity to any known proteins. This gene is expressed in TVCs,
endoderm, notochord and tail muscle (Fig.
4D). The other gene (ID 02049; DDBJ/EMBL/GENBANK accession number
AB125642), which encodes a Ca2+ transporting ATPase (ATP2A1/2/3),
is expressed in TVCs and tail muscle (Fig.
4E).
Cs-Mesp is essential for specification of TVCs
Cs-Mesp is the first gene among the four transcription factor
genes Cs-Mesp, NoTrlc, Cs-Nkx and Cs-HAND to be expressed in
the TVC lineage. Therefore, the function of Cs-Mesp was examined. To
suppress the function of Cs-Mesp, specific morpholinos were
microinjected into fertilized eggs. Embryos injected with one such morpholino,
Cs-Mesp-MO1, developed and hatched normally, similarly to control uninjected
embryos. The morpholino-injected larvae were also similar to controls in their
general morphology (Fig. 5A)
and swimming behavior. We confirmed normal differentiation of the endoderm
(Fig. 5B), epidermis
(Fig. 5C), nervous system
(Fig. 5D), mesenchyme
(Fig. 5E), notochord
(Fig. 5F) and muscle
(Fig. 5G) in the
morpholino-injected embryos by histochemical staining and whole-mount in-situ
hybridization of the marker genes.
|
|
Next, we examined whether TVCs retain their distinctive position despite losing expression of these TVC genes in the Mesp-knockdown embryo. The B7.5 cell of the morpholino-injected embryos was labeled with DiI at the 110-cell stage and the resultant embryos were observed under a fluorescent microscope. The B7.5 cell of the control embryo gave rise to TVCs and two muscle cells located in the anterior part of the tail (Fig. 1F, Fig. 4F). However, in the experimental embryo any B7.5-derived cells were not observed in the region where TVCs should be located (Fig. 4F'). Instead, four DiI-labeled cells were observed in the anterior part of the tail when Cs-Mesp function is suppressed. This suggests that B7.5 descendants with TVC fate would change their fate to that of embryonic muscle cells.
To examine the specificity of Cs-Mesp-MO1, another morpholino, designated
as Cs-Mesp-MO2, was designed to bind a different region of the Mesp
mRNA than does Cs-Mesp-MO1. Experiments using Cs-Mesp-MO2 yielded a similar
suppression of TVC-specific gene expression
(Table 1). These experiments,
in which two independent morpholinos for one gene gave the same result,
provide strong support for the specificity of these morpholinos
(Heasman, 2002).
In addition, we examined whether overexpression of Cs-Mesp mRNA leads to ectopic differentiation of TVCs. The microinjection of 60 pg of Cs-Mesp mRNA affected normal embryogenesis but did not induce any ectopic expression of HAND gene (n=7; data not shown), suggesting that Cs-Mesp alone is not sufficient for turning on the TVC program in cells other than the TVC lineage.
Cs-Mesp is essential for juvenile heart development
The failure of differentiation of the TVCs caused by Cs-Mesp
knockdown may lead to failure of differentiation of the juvenile heart after
metamorphosis. To test this possibility, we further examined Cs-Mesp
knockdown embryos, which hatch normally. The resulting larvae could swim and
then metamorphosed into juveniles, but all of the experimental organisms died
within 2 weeks after metamorphosis. As shown in
Fig. 6A, the heart in control
juveniles can be easily recognized as a tube-like structure due to the
transparency of the juvenile body (see Movie 1 at
http://dev.biologists.org/supplemental/).
By contrast, all of the experimental juveniles lacked a heart completely. That
is, in the experimental juveniles, no tube-like structure could be recognized
(Fig. 6A'; see Movie 2
http://dev.biologists.org/supplemental/)
(n=10 with Cs-Mesp-MO1 and n=9 with Cs-Mesp-MO2).
|
Forty-five percent of ß-catenin-morpholino-injected mid-gastrula embryos lacked the expression of Cs-Mesp, while the remaining embryos did express Cs-Mesp (Fig. 7B) (n=40). Cs-Mesp mRNA was not detected in Cs-macho1-morpholino injected embryos (Fig. 7C) (0%, n=24). These results were further confirmed by measuring the relative amounts of Cs-Mesp mRNA using real-time PCR. The quantities of Cs-Mesp mRNA in the ß-catenin- and Cs-macho1-morpholino-injected embryos were 37% and 2% of the quantity in control embryos, respectively (Fig. 7D). Therefore, both ß-catenin and Cs-macho1 are essential for Cs-Mesp transcription.
|
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Discussion |
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How are key genes such as Nkx and HAND regulated by
Mesp? Cs-Mesp expression terminates before the initiation of
the expression of Cs-Nkx, Cs-HAND and NoTrlc, although it
has not yet been determined how long Cs-Mesp protein is retained. Therefore,
Cs-Mesp may indirectly regulate the expression of Cs-Nkx, Cs-HAND and
NoTrlc. In the Cs-Mesp-suppressed embryos, cells fated to be
TVCs cannot migrate precisely to the ventral trunk region but migrate to the
tail region together with their sister cells fated to be anterior muscle
cells. In vertebrates, cells expressing Mesp genes receive positive
(BMP and FGF) and negative (Wnt) signals, and these signals activate
transcription of the key genes in the heart precursors
(Andree et al., 1998;
Reifers et al., 2000
;
Pandur et al., 2002
).
Similarly, Ciona TVCs may require such signals to express Cs-Nkx,
Cs-HAND and NoTrlc genes. In this case, the failure of migration
of the TVCs in the Cs-Mesp-suppressed embryos may disrupt
cellcell interaction between the TVCs and cells expressing such
signals, although it should be determined whether or not these signals are
also required for ascidian heart development. Two lines of experiments will
reveal the links between Mesp and the other key regulatory genes in
the ascidian embryo. One such line of experiments is analyzing the
cis-regulatory system of Nkx and HAND genes. Ciona
embryos provide an ideal system for this kind of assay
(Corbo et al., 2001
;
Satoh et al., 2003
) and,
actually, the cis-elements of NoTrlc have been analyzed in C.
intestinalis (Davidson and Levine,
2003
). The other line of experiments is identification of
Mesp-downstream genes. This can be easily realized on a genome-wide
scale, using a microarray covering almost all Ciona genes
(Azumi et al., 2003
). This
information will also illuminate the core system of vertebrate heart
development, which is expected to be conserved among chordates.
Genes specifying the ascidian endomesoderm
The organization of the ascidian embryonic endomesoderm is simple compared
with that of other chordates. In the trunk, endoderm is developed ventrally,
and TVCs and mesenchyme, including TLCs, differentiate in the lateral region.
In the tail, the axial notochord is flanked laterally by muscle cells. In
previous studies, we demonstrated that maternal ß-catenin is essential
for specification of this endomesoderm, except for muscle
(Imai et al., 2000). Maternal
ß-catenin, when it translocates from the cytoplasm to the nuclei of
vegetal blastomeres, activates key genes directly or indirectly, one or a few
of which is essential and sufficient for differentiation of each tissue. These
are Lhx3 for endodermal cells
(Satou et al., 2001a
),
Twist-like1 for mesenchymal cells
(Imai et al., 2003
) and
Brachyury for notochord cells
(Yasuo and Satoh, 1998
;
Corbo et al., 1997
). In the
present study, we demonstrated that Cs-Mesp is such a key gene for
specification of TVCs. While muscle cells are specified and determined by the
maternal macho1 gene, each of the endomesodermal tissues other than
muscle is specified through one zygotically expressed key gene. Therefore,
identification of upstream and downstream factors of these key genes will
reveal the complete gene circuits behind the ascidian larval endomesoderm
specification (Imai et al., submitted).
For understanding the entire genetic pathway from maternal information to
the final heart differentiation, it is also important to analyze the upstream
mechanism regulating the initiation of Cs-Mesp expression. In the
present study, we showed that both Cs-macho1 and ß-catenin are required
for the expression of Cs-Mesp. Cs-macho1 and ß-catenin are
thought to determine the posteriormost axis and vegetal axis, respectively.
The TVC precursors are located in the posteriormost and vegetal regions of the
embryo (Fig. 1). As expected,
transcription of Cs-Mesp is regulated by Cs-macho1 and
ß-catenin, although the ß-catenin knockdown is less
effective than the Cs-macho1 knockdown. Because the morpholino
against ß-catenin has been repeatedly used in previous studies
to confirm the suppression of its function effectively
(Satou et al., 2001b;
Imai et al., 2002a
;
Imai et al., 2002b
;
Imai, 2003
), the low
effectiveness may indicate that ß-catenin is not always essential for the
initiation of expression of Cs-Mesp but is required for reinforcement
or maintenance of the Cs-Mesp expression. The parental blastomere of
B7.5, i.e. B6.3, has developmental fates of germ cells, TVCs and muscle cells,
and the cell division at the 64-cell stage restricts one pair of its daughter
cells (B7.5) to the TVC and muscle fates (B7.5 is born at the 64-cell stage
and is retained at the 110-cell stage). Therefore, the most attractive
hypothesis is that release from germline repression initiates Cs-Mesp
expression at the 110-cell stage, as previously suggested by Davidson and
Levine (2003
). In this case,
Cs-macho1 and ß-catenin may directly activate Cs-Mesp.
The developmental fate of TVCs
It has been shown in another ascidian, H. roretzi, that
B7.5-derived cells give rise to latitudinal mantle muscle and atrial muscle in
addition to the heart in juveniles (Hirano
and Nishida, 1997). The adult body plan of Ciona is
somewhat different from that of Halocynthia
(Satoh, 1994
). They are
evolutionarily distant, because Halocynthia is an Enterogona ascidian
and Ciona is a Pleurogona ascidian, these two being major orders of
ascidians. Therefore, it should be determined whether the result of the
lineage trace experiments leading to that conclusion is also valid for
Ciona. For this purpose, we labeled a TVC precursor cell with DiI and
confirmed that TVCs give rise to the juvenile heart. However, we could not
observe DiI-labeling of latitudinal mantle muscle and atrial muscle in the
experimental juveniles. This is probably due to species differences, although
there is a possibility that it is due to the labeling method because Hirano
and Nishida (1997
) labeled the
blastomere with HRP. However, even if DiI-labeling was not as sensitive as
HRP, and Ciona TVCs also gave rise to these adult muscles, external
stimuli were found to cause our morpholino-injected animals to contract
normally, indicating that the mantle and atrial muscles had differentiated
normally even in the Cs-Mesp knockdown animals. Therefore, it is
highly likely that Cs-Mesp plays a specific role in heart
development.
Conclusion
As shown in the present study, ascidians have advantages for studying heart
development during chordate embryogenesis. The development of the heart in
ascidians appears to share a common mechanism with that in all chordates. The
specification of heart progenitors occurs in the 110-cell embryo, which
enables us to analyze the process at the single-cell level. The ascidian
genome contains fewer genetic redundancies, represented by the presence of
only one Mesp gene. Heart morphology can easily be observed because
the ascidian juvenile is transparent. Ascidian development is rapid, and thus
heartbeats can be observed within 3 days after fertilization. In addition, the
juvenile can survive for about 2 weeks without the heart. Therefore, the
ascidian provides a powerful system for studying chordate heart development
and this system may enable us to study the functions of genes that are
difficult to analyze in vertebrates.
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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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