Department of Biological Sciences, Graduate School of Science, Tokyo
Metropolitan University, 1-1 Minamiohsawa, Hachiohji, Tokyo 192-0397,
Japan
* Present address: Department of Zoology, Graduate School of Science, Kyoto
University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8502 Japan
Author for correspondence (e-mail:
saiga-hidetoshi{at}c.metro-u.ac.jp)
Accepted 9 September 2002
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SUMMARY |
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Key words: Halocynthia roretzi, Morpholino antisense oligonucleotide, Neural tube, Notochord, Zic family gene
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INTRODUCTION |
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The neural tube of ascidian larvae is composed of about 340 cells, and is
divided into three regions along the anteroposterior axis, which are, from
anterior to posterior, the sensory vesicle, the visceral ganglion and the
caudal neural tube (Nicol and
Meinertzhagen, 1991). The sensory vesicle is composed solely of
the a-line (anterior-animal) cells
(Nishida, 1987
). The visceral
ganglion present at the junction between the trunk and tail consists of the
A-line (anterior-vegetal) cells. The caudal neural tube running along the
length of the tail consists of four (dorsal, ventral and two lateral) rows of
ependymal cells: the lateral and ventral cells are of A-line origin and the
dorsal cells are of b-line (posterior-animal) origin. Beneath the neural tube,
a stack of exactly 40 notochord cells runs along the tail. The anterior 32
cells (primary notochord) and the posterior 8 cells (secondary notochord) are
derived from A-line and B-line cells, respectively
(Nishida, 1987
).
Cellular interactions that specify the neural tube and notochord of
ascidian embryos have been extensively demonstrated. Specification mechanisms
of neural tube differ between the a- and b-line precursors and A-line
precursors. The a-line neural tube precursors require an inductive influence
from the vegetal hemisphere cells to form the sensory vesicle
(Nishida and Satoh, 1989;
Okado and Takahashi, 1990
).
Upon disturbance of the induction, they adopt epidermal fate, like most other
animal hemisphere cells. Although timing of the induction has not been fully
understood, it is likely that the induction starts at the 16-cell stage and
becomes complete at the early gastrula stage, including multiple sequential
steps (Darras and Nishida,
2001b
; Nishida and Satoh,
1989
; Okado and Takahashi,
1990
). The b-line precursors also require an inductive influence
from the vegetal cells to differentiate into the caudal neural tube cells
(Hudson and Lemaire, 2001
). In
the induction of a- and b-line neural tube cells, an FGF-like signaling
pathway is likely involved, since human recombinant basic FGF mimics the
inductive activity of the vegetal hemisphere, and block FGF signaling leads to
inhibition of the sensory vesicle formation
(Darras and Nishida, 2001b
;
Hudson and Lemaire, 2001
;
Inazawa et al., 1998
;
Kim and Nishida, 2001
). This
situation is very reminiscent of neural induction in vertebrates. By contrast,
specification mechanisms of the A-line neural tube cells seems to be unique
(Minokawa et al., 2001
). At
the 32-cell stage, anterior-most A-line vegetal cells (A6.2 and A6.4
blastomere pairs) have both neural tube and notochord fates, which separate
into the daughter cells after the next cleavage. Anteriorly located daughters
succeed to the neural tube fate while posterior ones that contact the endoderm
precursors assume the notochord fate. A-line neural tube fate is specified
autonomously without any cellular interaction
(Minokawa et al., 2001
).
A-line notochord fate, however, requires inductive influence from the
endoderm precursors or the neighboring notochord precursors, which can be
mimicked by basic FGF (Darras and Nishida,
2001a; Kim and Nishida,
2001
; Nakatani and Nishida,
1994
; Nakatani et al.,
1996
; Shimauchi et al.,
2001
). Interestingly, all descendants of the isolated A6.2 or A6.4
blastomeres adopt the notochord fate when treated with basic FGF, and
conversely, they all adopt the neural tube fate in the absence of the
induction. Therefore, binary choice of the alternative fates is involved in
specification of the A-line neural tube and the notochord
(Minokawa et al., 2001
). In
contrast to our knowledge about cellular interactions involved in
specification of the neural tube, little is known about transcription factors
that participate in this process.
Zic was originally identified as a gene encoding zinc finger
protein that is expressed abundantly in the adult mouse cerebellum
(Aruga et al., 1994). In
vertebrates, multiple Zic family genes are known: for example, at
least six distinct Zic family genes have been identified in
Xenopus (Nakata et al.,
2000
) and at least four in mouse
(Aruga et al., 1996
).
Vertebrate Zic family genes so far identified are very similar to one
another both in structure and expression pattern during development
(Nagai et al., 1997
;
Nakata et al., 1998
). At the
gastrula stage, Zic family genes are expressed throughout the
presumptive neural plate. Their expression becomes restricted to the lateral
edges of the neural plate at the neurula stage and persists in the dorsal
region of the forebrain and the midbrain, the roof plate of the spinal cord,
the migratory neural crest and additionally in developing somites and limb
buds. The expression pattern of vertebrate Zic family genes suggests
their early roles in neural and neural crest development. In accordance with
this, overexpression experiments show that Xenopus Zic genes promote
differentiation of neural and neural crest derived tissues
(Nakata et al., 1998
). In
mouse, knockout of Zic1 leads to aplasia of cerebellum and skeletal
abnormalities (Aruga et al.,
1998
). Mutation in Zic2 and Zic3 in mouse and/or
human cause holoprosencephaly and heterotaxis, respectively
(Klootwijk et al., 2000
;
Nagai et al., 2000
). These
mutant phenotypes seem to be much weaker than expected from the results of
overexpression experiments.
In ascidians, macho-1, a recently identified muscle determinant,
encodes a zinc finger protein with the zinc finger domain most similar to that
of Zic family genes (Nishida and
Sawada, 2001). Transcripts of macho-1 are supplied to the
eggs maternally, segregated into the primary (the B-line) muscle cells through
rounds of cleavage, and have been shown to be necessary and sufficient for
primary muscle formation. Thus expression and function of macho-1 are
quite different from those of vertebrate Zic family genes. So far, no
ascidian Zic family gene other than macho-1 has been
reported.
In the present study, toward understanding of the neural development in ascidian embryos, we addressed whether there is another Zic family gene that may have a role in the neural development. We have cloned HrzicN, a new Zic family gene of the ascidian, Halocynthia roretzi and studied expression, function and regulation of HrzicN. We here report that HrzicN plays an essential role in neural tube development. Unexpectedly, we have also found that this gene is required for the formation of the notochord and anterior mesenchyme, representing a novel function of Zic genes. Thus, the present study has established that HrzicN is a key gene in the development of the two axial structures in the ascidian embryo.
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MATERIALS AND METHODS |
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Molecular cloning of HrzicN
PCR was carried out to amplify DNA fragments of a conserved region within
the zinc finger domain of Zic family genes. The nucleotide sequences
for forward and reverse primers were
5'-GCGAATTCTT(CT)AA(AG)GC(ACGT)AA(AG)TA(CT)AA-3' and
5'-CGCTGCAGTG(ACGT)AC-(CT)TTCAT(AG)TG(CT)TT-3', respectively. PCR
reaction was carried out through 40 cycles of denaturing at 94°C for 1
minute, annealing at 48°C for 1 minute and elongation at 63°C for 2
minutes using Halocynthia roretzi genomic DNA as template. The PCR
products were sequenced and two types of candidate clones for Zic
family genes were identified. One was of macho-1 and the other
represented a novel gene. Therefore, this was used as a probe for screening of
a neurula cDNA library. A cDNA clone was isolated and sequenced. 5'- and
3'-most regions for the cDNA were isolated by 5'- and
3'-RACE procedures, respectively, using the RACE System (Gibco BRL).
Construction of expression plasmids for in vitro transcription of
mRNA
To generate the expression plasmid for HrzicN, a full length
HrzicN cDNA was cloned into pBluescriptRN3
(Lemaire et al., 1995). To
generate the expression plasmid for lacZ (pRN3/lacZ), full length
lacZ of pSV-ß-Gal was cloned into pBluescriptRN3. To generate
the expression plasmid for HrzicN/lacZ, the 5' UTR and the
initiation codon of lacZ cDNA were substituted with the 5' UTR
and the first 183 bp of HrzicN coding region amplified by PCR. To
generate the expression plasmid for lacZ/HrzicN, the first 52nd base
pair of HrzicN cDNA was substituted with the first 123 bp of
lacZ cDNA. In vitro transcription was carried out using mMessage
mMachine (Ambion) as described previously
(Wada and Saiga, 1999b
).
Design of morpholino oligonucleotide and microinjection
Morpholino oligonucleotides were obtained from Gene Tools. Sequences of
HrzicNMO and HrzicNMO2 are
5'-GCTGTTGCGTATGCCATTTTTGCTT-3' (the underline
indicates sequence complimentary to the putative initiation codon) and
5'-ATTCGCTCAATTAAATTACTGTTGT-3', respectively. As a negative
control, `standard control oligo' supplied by Gene Tools was used.
Microinjection was carried out as described previously
(Wada and Saiga, 1999b).
Synthetic RNA and morpholino oligonucleotides to be injected were dissolved in
distilled water and 0.1x TE, respectively. Each microinjection
experiment was conducted twice or more.
To test the ability of HrzicNMO to inhibit translation, approximately 50 pg
of HrzicN/lacZ mRNA was injected into fertilized eggs either with or
without HrzicNMO (final concentration: 10 µM). Cleavage of the injected
embryos was inhibited by treatment with cytochalasin B
(Hirano et al., 1984). The
injected embryos were tested for ß-galactosidase activity at the middle
tailbud equivalent stage as described previously
(Hikosaka et al., 1994
).
For the rescue experiment, a mixture of HrzicNMO (final concentration: 10 µM) and approximately 50 pg of lacZ/HrzicN mRNA was injected into fertilized eggs and the injected embryos were examined for HrBra expression at the 110-cell equivalent stage.
Treatment with MEK inhibitor
To inhibit the FGF-Ras-MAP kinase signaling pathway, the MEK (MAP kinase
kinase) inhibitor (U0126, Promega) was used. Embryos were cultured in
Millipore-filtered seawater containing 2 µM U0126 as described previously
(Kim and Nishida, 2001).
Whole-mount in situ hybridization
Gene expression was visualized by whole-mount in situ hybridization as
described previously (Wada et al.,
1995).
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RESULTS |
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Expression pattern of HrzicN during embryogenesis
The spatial and temporal expression pattern of HrzicN during
larval development was examined by whole-mount in situ hybridization
(Fig. 2). As described below,
HrzicN expression was detected in the cells of neural and mesodermal
lineages from the early 32-cell stage to the neural plate stage.
|
Expression in the neural lineage
The larval ascidian neural tube originates from the a4.2, b4.2 and A4.1
cell pairs at the 8-cell stage. HrzicN expression was evident in all
three lineages of neural tube precursor cells. HrzicN expression was
first detected in A6.2 and A6.4 blastomere pairs, each of which contains both
neural tube and notochord fates, at the early 32-cell stage
(Fig. 2A,F,R). At the 44- and
64-cell stages, HrzicN expression was found in their daughter cells,
the A-line neural tube precursors (A7.4 and A7.8 pairs) and the A-line
notochord precursors (A7.3 and A7.7 pairs)
(Fig. 2B,C,G,H,R). The
expression continued in these cell lineages until the 110-cell stage
(Fig. 2D,E,I,J,R) but became
undetectable by the early gastrula stage
(Fig. 2K,P). At this stage,
however, HrzicN expression started in all the a- and b- line neural
tube precursors (a8.17, a8.19, a8.25, b8.17 and b8.19 pairs;
Fig. 2K,P). The expression
continued during gastrulation, disappeared by the early neurula stage
(Fig. 2L-N,Q) and no longer
detected afterwards (Fig.
2O).
Expression in the mesodermal lineage
As mentioned above, HrzicN was expressed in the primary notochord
lineage cells at the early 32-cell stage through the 110-cell stage. In
addition, HrzicN was expressed until the 110-cell stage in the B6.2
pair at the late 32-cell stage and their descendants
(Fig. 2R), which develop into
the secondary notochord, mesenchyme and primary muscle
(Fig. 2B-E,G-J,R). Like the
expression in the A-line cells, the expression in the B-line cells became
undetectable by the early gastrula stage
(Fig. 2K,P). In summary,
HrzicN expression was detected in all notochord precursors, one of
the two pairs of mesenchyme precursors and two out of the five pairs of
primary muscle precursors of the 110-cell stage embryo.
Phenotype induced by morpholino antisense oligonucleotide-based
translational inhibition of HrzicN
Recently, morpholino antisense oligonucleotide-based translational
inhibition was shown to be an effective tool for loss-of-function experiments
in the ascidian embryos (Satou et al.,
2001). We applied this technique to deduce functions of
HrzicN during embryogenesis. A morpholino oligonucleotide we prepared
(HrzicNMO) targets the initiation codon and its flanking regions. The ability
of HrzicNMO to inhibit translation was assessed by examining the effect of
HrzicNMO on translation of HrzicN/lacZ mRNA (a chimeric mRNA in which
the 5' UTR and the initiation codon of lacZ mRNA were
substituted with the 5' UTR and the first 183 nucleotides of
HrzicN mRNA coding region) in cleavage-arrested embryos. As
summarized in Table 1, HrzicNMO
was capable of disturbing translation of the mRNA with its target site.
|
To investigate the effect of translational inhibition of HrzicN mRNA on ascidian development, we injected HrzicNMO into fertilized eggs to achieve a final concentration of 1, 5 and 10 µM and reared them up to the swimming larva equivalent stage. As a negative control, we injected the `standard control oligo' supplied by Gene Tools and found that embryos injected at a final concentration of 10 µM or lower developed normally (Fig. 3C,D). Eggs injected with HrzicNMO at 1 or 5 µM developed into normal larvae (data not shown). However, almost all eggs injected with 10 µM developed into larvae with severe defects such as shortening of the tail, no differentiated notochord cells, failure of neural tube formation and lack of sensory pigment cells (Fig. 3B). However, protrusions, which are likely the adhesive organ, formed at the tip of the trunk (arrowhead in Fig. 3B). In the course of development, HrzicNMO-injected embryos seemed to be normal until early gastrula stage but delay in involution became evident in the later half of the gastrula stage (data not shown). The most notable abnormality was that they exhibited no sign of neurulation. Elongation of the tail was also inhibited, although a tail tip-like structure formed (Fig. 3A,B).
|
The phenotype generated by HrzicNMO injection seemed to be unique to
translational inhibition of HrzicN. First, an essentially identical
phenotype was observed upon injection of HrzicNMO2, another morpholino
nucleotide against HrzicN with a different and non-overlapping target
site from that of HrzicNMO (see inset in
Fig. 3B). This phenotype was
completely different from those observed upon injection of a morpholino
oligonucleotide against Hroth (S. W. and H. S., unpublished) or
ß-catenin mRNA (S. W., K. W. Makabe and H. S., unpublished). Second, to
verify specificity of effects of HrzicNMO, a rescue experiment was carried out
using lacZ/HrzicN mRNA, in which a translational initiation site was
provided by insertion of a lacZ fragment into the HrZicN
plasmid DNA so as to shift the translation initiation site 81 bp upstream to
the original initiation site of HrzicN. Thus, it is expected that
mRNA from this construct is free of translational inhibition by HrzicNMO,
since it has been shown that morpholinos that target more than a few bases
3' to the initiation codon exhibits a quite low efficiency
Summerton, 1999). Co-injection
of lacZ/HrzicN mRNA and HrzicNMO recovered HrBra expression
otherwise lost by injection of HrzicNMO
(Fig. 6B,G; for details about
marker gene expression in HrzicNMO-injected embryos, see below).
|
Injection of HrzicNMO into eggs at higher than 10 µM resulted in a phenotype similar to that obtained by injection at 10 µM (data not shown), so that injection at 10 µM seemed to be sufficient for inducing a representative phenotype by HrzicNMO. Therefore, we refer to embryos injected with HrzicNMO at 10 µM at the 1-cell stage as "HrzicN knockdown embryos".
Neural tube differentiation is disturbed in HrzicN knockdown
embryos
Since neurulation was blocked in HrzicN knockdown embryos, we
investigated the neural tube development in these embryos by analyzing
expression of neural markers. Initially, we assessed neural tube
differentiation in HrzicN knockdown embryos at the early tailbud
equivalent stage. First, we examined expression of a pan-neural marker,
HrETR-1 (Yagi and Makabe,
2001), which is expressed in the whole neural tube except for the
dorsal and ventral walls of the caudal neural tube, and the peripheral neurons
in the normal early tailbud stage embryo
(Fig. 4D). In HrzicN
knockdown embryos, weak HrETR-1 expression was found in superficial
cells of the trunk that seemed to be presumptive a-line neural tube cells, but
not in presumptive A-line neural tube precursors
(Fig. 4A). Next, we examined
expression of another neural marker, HrTBB2 (Halocynthia roretzi
ß-tubulin gene) (Miya and Satoh,
1997
), which is expressed in the neurons of the adhesive organ,
the neural tube in the trunk and peripheral epidermal neurons in the tail
(Fig. 4E). In HrzicN
knockdown embryos, expression of HrTBB2 was evident in the adhesive
organ-forming region and the epidermal neurons, but expression in the neural
tube-forming region was completely lost
(Fig. 4B). We examined
expression of HrTRP encoding the tyrosinase related protein
(Sato et al., 1999
) and
Hroth (Halocynthia roretzi otx gene)
(Wada et al., 1996
).
HrTRP is expressed in dorsal and lateral parts of the sensory vesicle
in the normal early tailbud stage embryo
(Fig. 4F), but it was not
expressed in HrzicN knockdown embryos
(Fig. 4C). Hroth
expression was also abnormal in HrzicN knockdown embryos. In the
normal early tailbud stage embryo (Fig.
4J) this gene is expressed in the sensory vesicle and the anterior
epidermis. In HrzicN knockdown embryos, Hroth expression was
lost from the sensory vesicle precursors, while it was detected only in the
anterior epidermis (Fig. 4G).
Together, these results indicate that differentiation of the neural tube is
severely affected in HrzicN knockdown embryos.
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Neural fate specification occurs in a- but not A-line precursors
In the a- and b-line precursors, HrzicN expression starts at the
early gastrula stage. Several lines of evidence suggest that fate choice
between epidermis and neural tube fates occurs earlier than this stage
(Darras and Nishida, 2001b;
Ishida et al., 1996
;
Yagi and Makabe, 2001
).
However, in the A-line neural tube cells, HrzicN expression starts at
the early 32-cell stage. Although it is unclear when the neural tube fate is
established in these A-line cells, the onset of HrzicN expression is
well before the appearance of neural properties such as HrETR-1
expression (this starts at the 110-cell stage). To see when abnormality in the
neural tube development arises in HrzicN knockdown embryos and
whether there is a difference in this process between a-line cells and A-line
cells, we next examined expression of these neural markers at earlier stages
of development. At the 110-cell stage, in control embryos, HrETR-1 is
expressed in the A-line neural tube precursors and only weakly in the a-line
neural tube precursors (Fig.
5F,G). At this stage, Hroth is also expressed in the
a-line neural tube precursors (Fig.
5H-J). In HrzicN knockdown embryos, HrETR-1
expression was evident in the a-line precursors but not in the A-line neural
tube precursors (Fig. 5A,B). The a-line precursors of these embryos were also positive for Hroth
expression (Fig. 5C-E). These
results suggest that initial specification of the neural tube precursors
occurs normally in a-line but not A-line precursors in HrzicN
knockdown embryos.
|
We then examined expression of the markers at the neural plate equivalent stage. At this stage, in control embryos expression of HrETR-1 and Hroth continues in the a- and A- line neural tube precursors and in the a-line precursors, respectively (Fig. 5Q,R). In the a-line precursors, HrTRP is also expressed (Fig. 5S). In HrzicN knockdown embryos, HrETR-1 expression was again absent from the A-line neural tube precursors (Fig. 5L) but evident in the a-line neural tube precursors, although the level of the expression was lower than that in control embryos (Fig. 5L,Q). Similarly, the expression of Hroth in the a-line neural tube precursors was reduced in HrzicN knockdown embryos (Fig. 5M,R). Furthermore, HrTRP expression was lost in them (Fig. 5N). These results suggest that the neural fate is once specified but not maintained at later stages of development in the a-line neural tube precursors.
Since early specification of the neural fate likely occurs in the a-line
neural tube precursors of HrzicN knockdown embryos, it is expected
that the epidermal fate is excluded from these cells. To test this
possibility, we first examined HrzicN knockdown embryos for
expression of HrEpiG (Ishida et
al., 1996), which occurs only in the epidermis precursors after
the 76-cell stage in normal development. HrEpiG expression was
normal, being excluded from both of the a- and b-line neural tube precursors
in HrzicN knockdown embryos at the early gastrula equivalent stage
(Fig. 5K,P). Next, we examined
HrzicN knockdown embryos at the early tailbud equivalent stage for
expression of HrEpiD (Ishida et
al., 1996
), which is another epidermis-specific gene expressed in
the whole epidermis except for a small area around the neuropore in the normal
embryo at this stage (Fig. 4K).
HrEpiD expression was excluded from a group of cells at the dorsal
side of HrzicN knockdown embryos. These cells were thought to be
descendants of the original neural tube precursors that had rejected the
epidermis fate (Fig. 4H). These
results indicate that the fate choice between neural tube and epidermis is
made successfully in HrzicN knockdown embryos.
Finally, we examined expression of HrzicN itself in HrzicN knockdown embryos. At the 110-cell stage, HrzicN expression in HrzicN knockdown embryos seemed to be identical to that in control embryos, indicating that HrzicN is not required for the maintenance of its early expression (data not shown). As mentioned previously, HrzicN expression disappears from the vegetal cells by the early gastrula stage in the normal development. By contrast, HrzicN expression was detected not only in the a- and b-line but also in the A-line neural tube precursors in HrzicN knockdown embryos at the middle gastrula equivalent stage (Fig. 5O,T). Thus, HrzicN expression in the A-line neural tube precursors failed to be suppressed in HrzicN knockdown embryos. Furthermore, HrzicN expression continued in dorsal superficial cells until early tailbud equivalent stage (Fig. 4I,L). From their position, these cells were thought to be descendants of the neural tube precursors that failed to form a neural tube. Thus, HrzicN expression in HrzicN knockdown embryos continued until a much later stage of development. This suggests that the activity of HrzicN is required for proper suppression of its own transcription.
Development of mesodermal tissues in HrzicN knockdown
embryos
As mentioned above, HrzicN was expressed in precursors for
mesodermal tissues, and HrzicN knockdown larvae exhibited a shortened
tail phenotype without differentiated notochord cells as judged by
morphological criteria. Therefore, we investigated development of the
notochord and other mesodermal tissues in HrzicN knockdown embryos by
examining marker gene expression. First we examined expression of
HrBra (Halocynthia roretzi Brachyury)
(Yasuo and Satoh, 1993) to
test whether the notochord fate was specified in HrzicN knockdown
embryos. Normally, HrBra is expressed in the A- and B-line notochord
precursors after the 64- and 110-cell stages, respectively
(Fig. 6F). We found
HrBra was not expressed in HrzicN knockdown embryos at the
110-cell stage (Fig. 6A) or at
the neural plate equivalent stage (data not shown). We also noticed another
abnormality in the gene expression profile of the notochord precursors in
HrzicN knockdown embryos. Expression of Hroth, which is
excluded from the notochord precursors in normal development
(Fig. 5H,I), was observed
ectopically in some notochord precursors in HrzicN knockdown embryos
(Fig. 5C,D). These suggest that
the notochord fate is not successfully specified in HrzicN knockdown
embryos.
HrzicN is also expressed in the anterior pair (B8.5 pair) of the two mesenchyme precursor pairs and two (B8.7 and B8.8 pairs) of the five muscle precursor pairs at the 110-cell stage (Fig. 2R). Therefore, expression of the muscle-specific actin gene was examined in HrzicN knockdown embryos at the 110-cell stage. In control embryos, all five pairs of the primary muscle precursors expressed the actin gene (Fig. 6J). This expression pattern was also observed in HrzicN knockdown embryos (Fig. 6E), suggesting that the muscle fate is properly specified in B8.7 and B8.8 pairs without HrzicN function.
To test this possibility further and to investigate development of the
mesenchyme precursors, we examined expression of Hrsna
(Halocynthia roretzi snail) (Wada
and Saiga, 1999a) at the 110-cell stage. Normally, Hrsna
is expressed in all notochord precursors, two A-line neural tube precursor
pairs (A8.15 and A8.16 pairs), both mesenchyme precursor pairs and all primary
muscle precursors (Fig. 6H,I).
In HrzicN knockdown embryos, Hrsna expression was lost from
all the notochord precursors, the two A-line neural tube precursor pairs
(A8.15 and A8.16 pairs) and the anterior mesenchyme precursor pair (B8.5 pair;
Fig. 6C,D). By contrast,
expression in the primary muscle precursors and the posterior mesenchyme
precursor pair (B7.7 pair) was unaffected
(Fig. 6C,D). This observation
strengthens the idea that the muscle fate is properly specified in B8.7 and
B8.8 pairs in HrzicN knockdown embryos. Also this result points to a
possibility that specification of the anterior mesenchyme precursor pair (B8.5
pair) requires HrzicN function.
In summary, these results suggest that among HrzicN-expressing cells, specification of all notochord precursors and anterior mesenchyme precursors was disturbed, while specification of muscle precursors was unaffected in HrzicN knockdown embryos.
HrzicN overexpression promotes neural development but not
notochord development
As described above, HrzicN seems to be essential for development
of the neural tube and notochord. To verify this idea, we next carried out
overexpression of HrzicN as a complementary experiment. Eggs were
injected with approximately 50 pg of HrzicN mRNA, cultured up to the
middle gastrula equivalent stage and examined for expression of marker genes.
We found that HrETR-1 was expressed in a half of the body of
HrzicN-overexpressing embryos. Judging from the size of cells, the
expression domain seemed to correspond to the animal hemisphere and its level
of expression was higher than that in control embryos
(Fig. 7A,B,D). Thus,
HrETR-1 expression was upregulated in HrzicN-overexpressing
embryos. This suggests that HrzicN promotes neural development by
activating downstream neural genes, directly or indirectly. However,
expression of HrBra was not detected in embryos injected with 50 pg
of HrzicN mRNA (Fig.
7C,E). Upon injection with approximately 5 or 15 pg of
HrzicN mRNA, HrBra expression was reduced as compared with
that in the control embryos (data not shown). Thus, HrzicN
overexpression affects development of the notochord and neural tube
differently. HrzicN alone may be insufficient to promote the
notochord fate. Alternatively, it is possible that the level of
HrzicN expression must be controlled precisely and/or that temporal
down-regulation of HrzicN expression may be important for this gene
to promote the notochord fate.
|
Regulation of HrzicN expression by FGF-like signaling
pathway
In ascidians, FGF-like signaling has been thought to be involved in
inductive interactions that are responsible for formation of the neural tube,
the notochord and the mesenchyme (Darras
and Nishida, 2001a; Darras and
Nishida, 2001b
; Hudson and
Lemaire, 2001
; Inazawa et al.,
1998
; Kim and Nishida,
2001
; Kim et al.,
2000
; Minokawa et al.,
2001
; Nakatani and Nishida,
1997
; Nakatani et al.,
1996
; Shimauchi et al.,
2001
). For example, treatment of embryos with a MEK inhibitor,
U0126, blocks formation of these tissues
(Darras and Nishida, 2001a
;
Kim and Nishida, 2001
). Since
our analyses so far showed significant correlation between HrzicN
expression and the neural tube, notochord and mesenchyme fates, we addressed
whether HrzicN expression is dependent on FGF-like signaling. Embryos
were treated with U0126 from the 1-cell stage onward and fixed at the 76-cell
stage or the middle gastrula equivalent stage to examine their HrzicN
expression as well as HrBra expression, which has been shown to
depend on FGF-like signaling as control
(Nakatani et al., 1996
).
HrzicN expression in the vegetal hemisphere at the 76-cell stage was
normal in U0126-treated embryos (Fig.
8A,E). By contrast, expression of HrzicN in the a- and
b-line neural tube precursors at the middle gastrula stage was inhibited in
U0126-treated embryos (Fig.
8B,F). As expected, HrBra expression was not detected in
U0126-treated embryos at both stages (Fig.
8C,D,G,H). These data indicate that HrzicN expression in
the vegetal cells is independent of FGF-like signaling, while HrzicN
expression in a- and b-line neural tube precursors depends on FGF-like
signaling.
|
![]() |
DISCUSSION |
---|
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---|
HrzicN is required for maintenance, but not for initial
specification, of neural tube fate in the a-line precursors
It has been shown that an inductive signal from the vegetal hemisphere
cells is required for formation of the a- and b-line neural tube
(Darras and Nishida, 2001b;
Nishida and Satoh, 1989
;
Okado and Takahashi, 1990
).
The induction likely occurs between the 16-cell and the early gastrula stage
and is mediated by FGF-like molecules
(Darras and Nishida, 2001b
;
Hudson and Lemaire, 2001
;
Inazawa et al., 1998
;
Kim and Nishida, 2001
;
Nishida and Satoh, 1989
). In
response to the induction, specification of the neural tube fate takes place
with activation of early neural genes such as HrETR-1 and
Hroth and suppression of the epidermis fate. HrzicN
expression starts in the a- and b-line precursors later than the onset of
expression of these genes and therefore, HrzicN may be unnecessary
for specification of the neural tube fate in these lineages. We further tested
this possibility by examining gene expression in HrzicN knockdown
embryos and found this is the case with the a-line precursors
(Fig. 9A). Furthermore, our
data suggest that HrzicN in a-line neural cells may be involved in
maintenance of the neural tube fate by keeping expression of early neural
genes active and by activating late neural genes such as HrTRP
(Fig. 9A). This view is
supported by the results of the overexpression experiment, showing that
HrzicN enhances HrETR-1 expression. It should be noted,
however, that some aspect of the genetic program for sensory vesicle formation
may be kept active in the knockdown embryo, because expression of
HrETR-1 did not vanish completely in the putative sensory vesicle
region of the knockdown embryo. Since the neural marker genes we used here are
not expressed in the b-line neural tube precursors, it was not determined
whether this is also the case with the b-line precursors.
|
In Xenopus, overexpression of any of the Zic family genes
caused transformation of the epidermis cells into neural and/or neural
crest-derived tissues (Mizuseki et al.,
1998; Nakata et al.,
1998
). Therefore, it has been thought that they are involved in
fate choice between epidermal and neural/neural crest fates. This seems to be
different from the function of the ascidian Zic suggested above. The
reason for this discrepancy is unknown but may be simply because
HrzicN and Xenopus Zic family genes play different roles
during neural fate specification, or it may be due to the difference in
methodologies or experimental systems. Further analysis of function of
vertebrate Zic genes and HrzicN may resolve this
problem.
HrzicN is necessary for specification of the neural tube
fate in the A-line precursors
The A-line neural tube cells are derived from A6.2 and A6.4 blastomeres of
the 32-cell stage embryo (Fig.
9B). In this lineage, unlike the a-line neural tube precursors,
HrzicN seems to be involved in the initial specification of the
neural fate, since HrETR-1 was not expressed in A-line precursors of
HrzicN knockdown embryos. Previous experiments showed that A6.2 and
A6.4 descendants adopt the neural tube fate autonomously without any cellular
interactions (Minokawa et al.,
2001). We have shown that vegetal expression of HrzicN is
independent of FGF-like signaling, a crucial and multifunctional regulator,
occurring around the onset of HrzicN expression. Therefore, a
possible model is that HrzicN expression is activated in the A6.2 and
A6.4 blastomeres autonomously and this in turn promotes initial specification
of the neural tube fate through activation of early neural genes like
HrETR-1 (Fig. 9B).
It has been shown that removal of the A-line neural tube precursors at the
64-cell stage leads to failure in pigment cell differentiation but not in
early neural fate specification in the a-line neural tube precursors
(Darras and Nishida, 2001b).
Therefore, it is possible that disturbance of the specification of the A-line
neural tube precursors may be the cause of defects in the development of the
a-line neural tube precursors in HrzicN knockdown embryos. However,
this cannot fully explain the a-line defects, because our preliminary
experiments showed that injection of HrzicNMO into a4.2 blastomeres of the
8-cell stage embryos leads to a failure in the sensory vesicle differentiation
similar to that shown in HrzicN knockdown embryos. Defects in
development of the a-line neural tube precursors found in HrzicN
knockdown embryos may occur as combined consequences of loss of
HrzicN function in both a- and A-line precursors.
HrzicN plays a novel role in notochord formation
We have shown that HrzicN is required for both of the primary and
secondary notochord cell formation. It has been shown that HrBra
expression in the notochord precursors and the notochord formation depend on a
cellular interaction with the endoderm precursors, which can be mimicked by
FGF (Darras and Nishida, 2001a;
Kim and Nishida, 2001
;
Nakatani and Nishida, 1994
;
Nakatani et al., 1996
;
Shimauchi et al., 2001
).
HrBMPb (Halocynthia roretzi BMP2/4) is also involved in this
process (Darras and Nishida,
2001a
). Together with the result that HrzicN expression
is independent of FGF-like signaling, we suggest that HrzicN is
required for A6.2, A6.4 and B6.2 blastomeres to respond to FGF-like molecules
(and/or HrBMPb) emanating from the endoderm precursors, and to activate
HrBra, which in turn promotes notochord development after the next
cleavage (Fig. 9B,C).
Previous reports showed that all descendants of the A6.2 and A6.4
blastomeres assume the neural fate when they are in isolation and, conversely,
they adopt the notochord fate after treatment with human recombinant basic FGF
(Minokawa et al., 2001;
Nakatani and Nishida, 1994
;
Nakatani et al., 1996
). Since
HrzicN is required for the A6.2 and A6.4 blastomeres to develop into
both the neural tube and the notochord, it is possible that HrzicN
prompts these blastomeres to pursue the neural fate in the absence of FGF-like
signaling while it allows them to follow the notochord fate in the presence of
the FGF-like signaling.
Developmental fate of the A6.2 and A6.4 blastomeres in HrzicN knockdown embryos is unclear. It is unlikely that they adopt the epidermis fate because expression of HrEpiG was excluded from them. It is also unlikely that they assume the endoderm fate because endoderm-specific alkaline phosphatase activity was restricted to the original endoderm cells in HrzicN knockdown embryos (data not shown). One possibility is that they remain undifferentiated, although expression of other markers must be examined to verify this possibility.
HrzicN is required for specification of the anterior
mesenchyme but not for the primary muscle
It has been shown that the B6.2 blastomere requires FGF-like signaling from
the endoderm precursors to form not only the notochord but also the mesenchyme
(Kim and Nishida, 1999;
Kim and Nishida, 2001
;
Kim et al., 2000
). In the
absence of the signaling, all of B6.2 descendants pursue muscle fate possibly
because of the action of muscle determinants they inherit
(Kim and Nishida, 1999
;
Kim and Nishida, 2001
). We
found that HrzicN knockdown led to failure in specification of B8.5
blastomeres into mesenchyme. In this case, the blastomeres did not exhibit a
muscle character either. Together with a role of HrzicN in the
notochord specification in B8.6 blastomeres discussed above, we suggest that
HrzicN is required for the B6.2 blastomeres to respond to FGF-like
molecules to facilitate the notochord and the mesenchyme fates. Suppression of
the muscle fate probably occurs independently of HrzicN
(Fig. 9C).
Expression of the actin gene and Hrsna was normal in two primary
muscle precursor pairs, the B8.8 and B8.7 pairs, irrespective of
HrzicN expression there. This indicates that HrzicN is
unnecessary for these cells to adopt muscle fate. It is well known that the
primary muscle of ascidian embryos develops autonomously owing to the action
of maternally provided cytoplasmic determinants. Recently, macho-1, a
muscle determinant has been isolated from Halocynthia roretzi
(Nishida and Sawada, 2001).
Depletion of the transcripts results in loss of all of the primary
muscle cells and overexpression of macho-1 caused ectopic muscle
formation in non-muscle-lineage such as endoderm and epidermis. Therefore, it
is highly likely that HrzicN is not required for primary muscle
development: it is dependent on macho-1
(Fig. 9C). However, since both
macho-1 and HrzicN belong to the Zic family, they
may interact to provide the B8.8 and B8.7 pairs with unknown specific
characters. Such a possibility should be tested in future study.
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ACKNOWLEDGMENTS |
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