Unité de Biologie du Développement (UMR7009), CNRS/UPMC, Station Zoologique, Observatoire Océanologique, 06230 Villefranche-sur-mer, France
e-mail: clare.hudson{at}obs-vlfr.fr and yasuo{at}obs-vlfr.fr
Accepted 5 January 2005
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SUMMARY |
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Key words: FGF, Nodal, ALK4/5/7, Ciona, Neural patterning, Secondary muscle, Ascidian, Tunicate
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Introduction |
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We have been using these simple embryos to study development and patterning
of the central nervous system (CNS). The ascidian larval CNS consists, along
the anterior-posterior axis, of a sensory vesicle, neck, visceral ganglion and
tail nerve cord (Fig. 1) (for
reviews, see Lemaire et al.,
2002; Meinertzhagen and
Okamura, 2001
; Meinertzhagen
et al., 2004
). The entire CNS consists of only around 330 cells.
The simplicity of the ascidian neural tube is most apparent at the level of
the tail nerve cord, which is only four cells in cross section, one dorsal
(b-line), two lateral and one ventral (A-line). Despite this comparative
simplicity, the ascidian CNS retains many features in common with its
vertebrate counterpart. The expression of many genes along the dorsal-ventral
and anterior-posterior axes of the CNS is conserved between ascidian and
vertebrate embryos. Orthologues of Otx, Pax2/5/8 and Hox genes are
differentially expressed along the anterior-posterior axis, while genes
encoding Hedgehog and HNF3ß are expressed in the
ventral neural tube and Snail, Pax3/7, BMP2/4 and Msx are
expressed in the lateral or dorsal part of the neural tube
(Aniello et al., 1999
;
Corbo et al., 1997a
;
Hudson and Lemaire, 2001
;
Imai et al., 2002
;
Miya et al., 1997
;
Takatori et al., 2002
;
Wada et al., 1997
;
Wada et al., 1998
;
Wada and Saiga, 1999a
;
Wada and Saiga, 1999b
).
Patterning of both the ascidian and vertebrate CNSs along the
anterior-posterior and dorsal-ventral axes starts during gastrulation when the
CNS exists as a neural plate.
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The mechanisms underlying the specification of neural fate in ascidian
embryos have begun to emerge. The onset of acquisition of neural fate in the
a- and b-lineages involves induction by one of the fibroblast growth factor
family members, Ci-FGF9/16/20 (Bertrand et
al., 2003). In the absence of induction by Ci-FGF9/16/20, these
cells adopt an epidermal fate (Bertrand et
al., 2003
). By contrast, neural fate in the A-line is specified
following a cell-fate decision between notochord and neural fates in which the
neural fate is adopted in a cell-autonomous manner
(Minokawa et al., 2001
).
While the specification of neural fate in ascidians is reasonably well
understood, very little is known about how the neural lineages become
patterned. The Ras/MEK/ERK/Ets signalling pathway, which is activated
downstream of FGF-like signalling, has been implicated in posteriorisation of
the neural tube (Akanuma and Nishida,
2003; Hudson et al.,
2003
). In embryos in which this signalling pathway is inhibited,
markers of posterior neural fate are lost and a greater number of A-line cells
express markers of the anterior CNS.
In this study, using blastomere ablation experiments and analysis of the FGF and Nodal signalling pathways, we addressed how the ascidian neural plate becomes patterned across the medial-lateral (future ventral-dorsal) axis, mainly focusing on the A-line neural lineages.
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Materials and methods |
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Embryo culture and manipulation
Embryo culture and cytochalasin (Sigma) and U0126 (Calbiochem) treatments
have been described previously (Hudson et
al., 2003). SB431542 was purchased from Tocris and used at a
concentration of 5 µmol/l, which was the lowest concentration giving robust
inhibition of Ci-Snail and Ci-Delta2 expression at the early
gastrula stage. SB431542 was added to embryos at the 16-cell stage, just prior
to the onset of Ci-Nodal expression at the 32-cell stage, until
fixation, except for embryos in Fig.
6E,F, where SB431542 was washed away at the mid-gastrula stage
when Ci-Nodal expression is no longer detectable in b-lineages.
Unfertilised eggs were microinjected as described previously
(Hudson et al., 2003
). The
concentration chosen for injection of mRNA or morpholino was the lowest
concentration giving a consistent larval phenotype and effect on
Ci-Delta2 and Ci-Snail expression at early gastrula stage.
This was 0.25 µg/µl for Ci-tALK4/5/7 mRNA, 0.125 µg/µl
for Ci-Nodal mRNA and 0.4-0.5 mmol/l for Nodal morpholino.
Ci-FGF9/16/20 morpholino was injected at 0.25 mmol/l. Blastomere ablation was
carried out by microinjecting the blastomere with water until it burst.
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Results |
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To address whether signals from b6.5 are required to pattern the adjacent A-line neural blastomeres, we ablated the b6.5 blastomere on the right side at the 32-cell stage, leaving the left side as a control. We then analysed expression of Ci-Otx at the neurula stage in embryos that had been treated with cytochalasin B from the 64-cell stage to facilitate analysis (Fig. 2A). In the presence of cytochalasin B, cytokinesis is blocked, but blastomeres continue to express marker genes consistent with the principal fates that they would adopt during normal development. In unoperated embryos, Ci-Otx is expressed in the A7.4 blastomere but not in the A7.8 blastomere. However, following ablation of b6.5, expression of Ci-Otx was also observed in the A7.8 blastomere on the right side. Thus, ablation of the b6.5 blastomere affected patterning of the A-line neural lineages.
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Taken together, these results show that the b6.5 blastomere is required to signal to the lateral A7.8-line cells to instruct them to undertake a different developmental programme to that of the medial A7.4-line cells. We next investigated the nature of this instructive signal, which should originate from the b6.5 blastomere and be dependent upon MEK signalling.
Ci-Nodal expression is induced in b6.5 blastomeres by Ci-FGF9/16/20
Activation of ERK is seen in the b6.5 blastomere during the late 32-cell
stage (Hudson et al., 2003;
Nishida, 2003
). We have found
that a Ciona homologue of the TGFß family member,
Ci-Nodal, starts to be expressed in this blastomere at the same stage
(Fig. 3A). At the 32-cell
stage, Ci-Nodal expression is also transiently observed in the
vegetal cells A6.1, A6.3 and B6.1 (Fig.
3A). Expression of Ci-Nodal in b6.5 is maintained in its
descendants during the following cell divisions until mid-gastrula stages
(Fig. 3B) (C.H. and H.Y.,
unpublished) (Imai et al.,
2004
; Morokuma et al.,
2002
). Thus, Ci-Nodal is expressed in the b6.5 blastomere
and at the right time, making it a good candidate for the signal required to
pattern the A-line neural lineages.
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Ci-FGF9/16/20 is broadly expressed in the vegetal hemisphere of
embryos from the 16-cell stage and has recently been shown to be required for
Ci-Otx expression in a6.5 and b6.5 at the 32-cell stage
(Bertrand et al., 2003). We
therefore addressed whether Ci-FGF9/16/20 is required for Ci-Nodal
expression in b6.5. In embryos injected with antisense morpholino
oligonucleotides against Ci-FGF9/16/20 (Ci-FGF9/16/20-MO), Ci-Nodal
expression was no longer observed in the b6.5 blastomere
(Fig. 3A). As with U0126
treatment, Ci-FGF9/16/20-MO injection reduced, but did not eliminate,
Ci-Nodal expression in the vegetal cells
(Fig. 3A).
Finally, if Ci-Nodal in b6.5 is the signal that patterns the A-line neural cells, Ci-Nodal expression should become independent of MEK signalling by the end of the 32-cell stage, that is, when patterning of the A-line neural lineages with respect to Ci-Otx expression becomes independent of MEK. To test this we treated embryos with U0126 from the early 32-cell stage or from the late 32-cell stage and analysed expression of Ci-Nodal at the early gastrula stage, when Ci-Nodal was expressed in the four b6.5 derivatives b8.20, b8.19, b9.18 and b9.17 (Fig. 3B). We found that Ci-Nodal expression became independent of MEK activity by the end of the 32-cell stage (Fig. 3B).
These results indicate that Ci-FGF9/16/20 signalling is required for activation of Ci-Nodal expression in the b6.5 blastomere at the 32-cell stage. Ci-Nodal is thus an excellent candidate for the signal responsible for patterning of the A-line neural lineages.
Nodal signals are required for patterning of the A-line neural lineage, but not for specification of neural fate
In order to investigate the role of Nodal during neural patterning, three
methods were chosen to inhibit Nodal signalling. Firstly, we used a
pharmacological inhibitor, SB431542, which blocks the TGFß type I
receptors ALK4, ALK5 and ALK7, for Activin and Nodal ligands, without
inhibiting other ALK family members that bind to BMP ligands
(Inman et al., 2002). In the
Ciona genome, one potential Nodal receptor is apparent, named
Ci-TGFß-receptor Ic, which appears to represent
vertebrate ALK4, -5 and -7 and thus in this study
we refer to it as Ci-ALK4/5/7
(Hino et al., 2003
). As a
second method to inhibit Nodal signalling, we constructed a truncated version
of Ci-ALK4/5/7 in which the cytoplasmic domain was removed. This
truncation of the type I receptors of the TGFß ligand superfamily has
been shown in other systems to act as a dominant negative
(Chang et al., 1997
;
Suzuki et al., 1994
). Finally,
we injected an antisense morpholino oligonucleotide against Ci-Nodal
(Ci-Nodal-MO) to knock down the Nodal ligand. Analysis of the Ciona
genome has revealed the presence of a single Nodal gene
(Hino et al., 2003
).
Using these three reagents to inhibit Nodal signalling, we looked at expression of Ci-Snail and Ci-Delta2 at the early gastrula stage. Inhibition of Nodal signalling by all three methods abolished Ci-Snail and Ci-Delta2 expression in A8.15/A8.16 (Fig. 4A; Table 1). Expression of Ci-Delta2 in the b-line cells was also lost, suggesting that Ci-Nodal may also play a role in b-line fate (Fig. 4A; Table 1). Expression of Ci-Snail in the primary muscle lineages was not affected. In contrast to inhibition of Nodal, ectopic activation of Nodal signalling by injecting Ci-Nodal mRNA into eggs had the opposite effect, such that Ci-Delta2 and Ci-Snail were expressed in up to all eight A-line neural cells in 59% (16/27 in all eight cells) and 53% (19/36 in all eight cells) of cases, respectively (Fig. 4A).
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These results show that Nodal signalling is required for the lateral A-line neural precursors to initiate a molecular programme different from that of the medial precursors, but is not required for the formation or maintenance of generic neural fate.
A collection of neural plate markers to investigate neural patterning
The neural plate of ascidian embryos exhibits a highly regular organisation
of cell arrangement and cell division pattern. At the mid-gastrula stage,
before the neural plate starts to roll up, the neural plate consists of six
rows of a total of 44 neural cells aligned as shown in
Fig. 5 (top). A-line neural
lineages contribute to the first two caudal rows, while a-line neural lineages
form the remaining four rows. Three cells of the b-line neural lineages flank
each side of the neural plate. In order to investigate in more detail the
consequences of Nodal inhibition on neural plate patterning, we first
conducted a small-scale in situ hybridisation screen to collect genes
expressed in a distinct manner in the neural plate. Six markers collected
during the course of the screening are shown in
Fig. 5. Expression patterns of
the markers are shown in embryos in which the neural plate consists of six
rows of cells and the following stage when the cells in row II of the neural
plate have divided along the rostral-caudal axis, resulting in a neural plate
of seven rows. Ci-HES-b is expressed in the lateral cells of the
A-line neural plate (A9.32, A9.30, A9.29), including more medial A7.4
derivatives (A9.15, A9.16) and row III a-line neural plate (a9.49, a9.33)
(Fig. 5A). Ci-Chordin
is expressed in the lateral-most A- and a-line blastomeres of all six rows of
the neural plate (A9.32, A9.30, A9.29, a9.49, a9.50, a9.51, a9.52), as well as
the b-line neural cells, epidermal cells bordering the neural plate and weakly
in the notochord (Fig. 5B).
More specific markers of the lateral A-line neural lineage are
Ci-Lefty, which is expressed in A9.29, and Ci-FGF8/17/18,
which is expressed in A9.30 (Fig.
5C,D). Ci-Lefty is also expressed in the posterior
epidermis with variable intensity. Markers of medial A-line neural cells are
Ci-HB9/MNX and Ci-FGF9/16/20. Ci-HB9/MNX is expressed in
A9.15 and A9.13 at the six-row neural plate stage, becoming weaker in A9.15 at
the seven-row neural plate stage (Fig.
5E). During tailbud stages, expression of Ci-HB9/MNX
reappears in the CNS and is found in the motoneurons, which are a lateral
A-line neural cell derivative (see below). Ci-FGF9/16/20 is expressed
in A9.16 and A9.14 at the six-row neural plate stage and also in a9.34 and
a9.38 at the seven-row neural plate stage
(Fig. 5F). Both these latter
genes are also expressed in the primary muscle lineages. Using these markers,
we investigated the effects on the patterning of the neural plate when Nodal
signalling was perturbed.
Lateral neural tissue and secondary muscle markers are not expressed following Nodal inhibition
We tested expression of all markers in embryos treated with SB431542 and
Nodal-MO injection and examples of both are shown in Figs
6 and
7. Some markers were also
tested in embryos injected with Ci-tALK4/5/7 mRNA
(Table 2;
Fig. 7B). In all cases,
equivalent results were obtained (Figs
6,
7;
Table 2).
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We also addressed whether a specific neuronal type derived from the lateral
neural plate was generated in the absence of Nodal signals. Motoneurons of
ascidian embryos originate from the A8.15 lineage
(Fig. 1)
(Cole and Meinertzhagen, 2004).
Interestingly, it has been observed that motoneurons may still form in
isolated A4.1 explants (Okada et al.,
1997
). We used two motoneuron markers, Ci-ChAT, which
encodes cholinergic acetyltransferase
(Takamura et al., 2002
;
Yoshida et al., 2004
), and the
Ciona homologue of HB9 and MNR2 genes, which play a
crucial role in motoneuron specification in other systems, and of which there
is one representative in the Ciona genome, Ci-HB9/MNX
(Broihier and Skeath, 2002
;
Odden et al., 2002
;
Shirasaki and Pfaff, 2002
;
Wada et al., 2003
). Expression
of these motoneuron markers was completely dependent on an intact Nodal
signalling pathway (Fig. 6E,F;
Table 2). Therefore, the
formation of these specific neuronal cell types also depends upon Nodal
signalling.
The A8.15/A8.16 blastomeres generate the secondary muscle lineage as well
as the lateral neural plate. Formation of the so-called secondary muscle
requires inductive interactions, unlike the autonomously determined primary
muscle (Meedel et al., 1987;
Meedel et al., 2002
;
Nishida, 1990
). At the
110-cell stage, the A7.8 blastomere divides to generate A8.15 (neural fate)
and A8.16 (neural and muscle fates) (Fig.
1). Finally, A8.16 divides into muscle (A9.31) and neural (A9.32)
precursors during neural plate stages (Fig.
1) (Cole and Meinertzhagen,
2004
; Nicol and Meinertzhagen 1998a; Nicol and Meinertzhagen
1998b; Nishida, 1990
). As the
A9.31 secondary muscle comes from the same lineage as the lateral neural
plate, we investigated whether the secondary muscle lineage was also affected
by Nodal inhibition. In embryos that had been cleavage arrested after the
76-cell stage when A7.8 had divided into A8.15 and A8.16, expression of
Ci-Actin could be detected in the secondary muscle lineage (A8.16),
which remained in a different position to the primary muscle lineage and could
thus be easily distinguished (Fig.
6G, arrowheads). Formation of secondary muscle was inhibited in
embryos in which Nodal signalling was blocked
(Fig. 6G;
Table 2). This suggests that
the entire lineage of the A8.15/A8.16 blastomere, which gives rise to lateral
neural tube and secondary muscle, requires Nodal signals for its fate
specification.
Medial A-line neural plate fates expand following Nodal inhibition
We next addressed whether the inhibition of Nodal causes a general
disruption of neural plate patterning, or affects only the lateral neural
plate fates. We analysed expression of Ci-HB9/MNX and
Ci-FGF9/16/20, which are expressed in distinct sets of medial A-line
cells of the neural plate (Figs
5,
7). In Nodal-inhibited embryos,
these markers continued to be expressed in the correct row of neural plate
cells, but their expression was expanded laterally to a maximum of eight cells
in total (Fig. 7A,B). We also
analysed Ci-Otx expression at neurula stages in embryos
cleavage-arrested in cytochalasin B from the 64-cell stage. Under these
conditions, Ci-Otx was expressed only in the medial A7.4 blastomeres
of otherwise unmanipulated embryos. When Nodal signalling was blocked,
however, Ci-Otx was also expressed in the lateral A7.8 cells
(Fig. 7A). Altogether, this
suggests that, within the A-line neural lineages, Nodal signalling
specifically determines lateral cell fates, and that, in the absence of Nodal,
the lateral cells adopt a medial-cell-like fate.
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Discussion |
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Our observations suggest that the action of Ci-Nodal on the lateral A-line
neural precursors probably occurs directly, rather than via activation of
other signalling pathways in surrounding cells. Firstly, the cell ablation
studies indicate that the signal from b6.5 is occurring during the 32-cell
stage, when these two cell types are in direct contact. Secondly, injection of
Ci-Nodal mRNA revealed that the medial A-line neural precursors are
capable of responding to Nodal and expressing lateral markers at the early
gastrula stage. This suggests that, during normal embryogenesis, the medial
A-line neural precursors do not express lateral markers because they are not
in contact with b6.5 and therefore do not receive a Nodal signal. Furthermore,
it is unlikely that neural plate patterning defects are a consequence of a
general perturbation of cell fate specification, because treatment of embryos
with SB431542 does not affect expression of markers for primary notochord
[Ci-Bra at the 64- and 110-cell stages
(Corbo et al., 1997b);
Ci-Chordin in Fig.
6A], primary muscle (Ci-Snail in
Fig. 4 and Ci-Actin in
Fig. 6), or endoderm
[Ci-Titf1 at the early gastrula
(Ristoratore et al., 1999
)]
(C.H. and H.Y., unpublished).
In addition to mediating A-line neural patterning, Nodal signals were also required for expression of lateral neural fate markers in the a- and b-line neural lineages, as seen by the loss of Ci-Delta2 (b-line), Ci-Chordin (a- and b-line) and Ci-HES-b (a-line) expression following Nodal inhibition (Figs 4, 5, 8). This suggests that Nodal signalling is required for lateral patterning of the entire neural plate.
Distinct mechanisms control anterior-posterior and medial-lateral patterning of the neural plate
We have found that Nodal signalling is involved in medial-lateral
patterning of the neural plate but not in anterior-posterior patterning.
Ci-HB9/MNX, Ci-FGF9/16/20 and Ci-Otx are differentially
expressed along the anterior-posterior axis of the neural plate in row I, row
II and rows III-VI, respectively. In embryos in which Nodal signalling was
blocked, the expression domains of these genes remained in their correct
anterior-posterior positions (Fig.
4B, Fig. 7). Therefore, the mechanisms leading to anterior-posterior and medial-lateral
patterning of the neural plate are separable. There are some indications that
the MEK signalling pathway may play a role in anterior-posterior patterning of
the neural plate in addition to its role in medial-lateral patterning via
activation of Ci-Nodal. For example, Ci-HB9/MNX is not
expressed in the neural plate of embryos treated with an inhibitor of MEK,
while this gene continues to be expressed in the correct row of cells
following Nodal inhibition (Hudson et al.,
2003) (present study).
Nodal signalling and secondary muscle formation
In ascidians, muscle cells originate from three lineages. The primary
lineage derives from the B-line and is specified cell-autonomously (e.g.
Nishida and Sawada, 2001). The
secondary lineage arises from the b6.5- and A8.16-lineages
(Nishida, 1987
;
Nishida, 1990
). In this study,
we observed that the formation of the A8.16-derived secondary muscle lineage,
like lateral neural fates, depended on Nodal signals. In contrast to the
primary lineage, the secondary lineage is known to require cellular
interactions for fate specification; isolated A4.1 blastomeres of the 8-cell
stage embryo do not form muscle fate unless recombined or co-isolated with
animal blastomeres (Meedel et al.,
1987
; Meedel et al.,
2002
; Nishida,
1990
). In addition, it has been shown in Halocynthia that
FGF/Ras/MEK signalling is required between the 32- and the 64-cell stage for
secondary muscle cell formation (Kim and
Nishida, 2001
). We propose that the role of FGF signalling in
secondary muscle formation is indirect, via the activation of
Ci-Nodal in b6.5 blastomeres. The A8.16 blastomere forms following
cell division of A7.8 in the medial-lateral direction and remains in contact
with the Ci-Nodal expressing b6.5 descendants. A8.15, the sister
blastomere of A8.16, is positioned more medially, such that it is no longer in
contact with the b6.5 descendants. The A8.15 blastomere gives rise to only
neural fates, whereas the A8.16 blastomere gives rise to neural and secondary
muscle fates. It is possible that a short exposure to Nodal signals results in
lateral neural fate, the fate taken by A8.15, and a longer exposure is
required for induction of muscle fate in the A8.16 lineage. Consistent with
this idea, in Halocynthia, A8.16 blastomeres isolated early in their
cell cycle do not form muscle, whereas some of those isolated late in their
cell cycle can develop autonomously into muscle
(Nishida, 1990
).
Nodal signalling during development of ascidians and vertebrates
During development of vertebrate embryos, Nodal signals are involved in
formation of the anterior-posterior and left-right axes and specification of
the endoderm and mesoderm germ layers
(Schier and Shen, 2000;
Whitman, 2001
;
Bertocchini and Stern, 2002
;
Perea-Gomez et al., 2002
). Its
role in left-right axial patterning appears to be conserved between ascidians
and vertebrates (Morokuma et al.,
2002
). However, we did not observe profound effects following
inhibition of Nodal signals on expression of marker genes for endoderm and
mesoderm. It is possible that, due to the rapid development of ascidian
embryos, the role of Nodal in endoderm and mesoderm has been bypassed by the
recruitment of maternal determinants to specify the major tissue types.
Alternatively, since Nodal signalling is not involved in germ layer
specification in echinoderms, this role may be a vertebrate invention
(Duboc et al., 2004
). Nodal
signalling is required for secondary muscle induction in Ciona,
suggesting that at least some mesoderm cell-types are dependent on Nodal
signalling. In addition, Ci-Nodal is expressed in the vegetal cells
at the 32-cell stage, albeit transiently, and embryos do not gastrulate
correctly following inhibition of Nodal signalling. Therefore, there are
likely to be further roles for Nodal signalling during ascidian development
that remain to be understood.
The vertebrate neural tube is patterned across the dorsal-ventral
(lateral-medial neural plate) axis, by signals from tissues adjacent to, as
well as from within, the neural tube. A variety of signalling molecules has
been identified in this patterning event. Formation of the floor plate, the
ventral-most part of the spinal cord, involves Nodal and SHH signalling
pathways (for reviews, see Appel,
2000; Lewis and Eisen,
2003
; Strahle et al.,
2004
). In zebrafish embryos, Nodal signalling is required for
specification of floor plate precursors. In mouse and chick embryos, however,
SHH signalling appears to play a more pivotal role during floor plate
specification, with the role for Nodal much less clear, although recently,
roles for Nodal have begun to emerge in these vertebrates as well
(Lewis and Eisen, 2003
;
Strahle et al., 2004
).
Subsequent to floor plate formation, SHH signalling from the floor plate and
notochord patterns the ventral part of spinal cord to specify distinct
neuronal precursors. In contrast, the dorsal part of the neural tube is
patterned by BMP signalling from the laterally (future dorsally) situated
epidermis that borders the neural plate and later from the roof plate
(reviewed by Altmann and Hemmati-Brivanlou,
2001
; Lee and Jessell,
1999
).
In this study, we uncovered a number of differences in the role of Nodal signalling in ascidian neural patterning compared with vertebrates. Firstly, in ascidians, Nodal ligands emanate from cells laterally flanking the neural plate, not from axial tissues as in vertebrates. Secondly, Nodal signals are required for lateral neural fates, but not for medial (future ventral) fates. Finally, Nodal signals in ascidians restrict medial neural fates by promoting lateral fates. It may be that in the ancestral chordate Nodal was involved in both dorsal and ventral neural fate specification. In this case, one could postulate that the role of Nodal during induction of lateral fates has been lost (or perhaps overlooked) in vertebrate lineages, and the role of Nodal during formation of ventral fates has been lost in ascidians. Alternatively, the role of Nodal signalling in dorsal or ventral patterning may have been recruited independently in vertebrate and invertebrate chordates, respectively. It will be important to look again in vertebrates to see if the role of Nodal signalling during dorsal patterning of the neural tube has been overlooked owing to the severe developmental defects that occur earlier during germ layer formation upon Nodal inhibition.
Despite this apparent difference in the role of Nodal signalling during CNS
patterning, other aspects of dorsal-ventral neural tube patterning may be
conserved. For example, an ascidian homologue of hedgehog, Ci-hh2, is
expressed in the ventral-most cell of the tail nerve cord from the early tail
bud stage and BMP2/4 is expressed in the borders of the neural plate
(Miya et al., 1997;
Takatori et al., 2002
).
Furthermore, BMP2/4-Chordin antagonism is required for formation of the
pigment cells, a dorsal cell fate derived from the a-line lateral neural
plate, implicating this pathway in dorsal patterning
(Darras and Nishida, 2001
).
Future work should study the relationship between Nodal, SHH and BMP2/4
signalling pathways. It will also be important to determine whether the medial
neural fate of the ascidian neural plate is an induced fate or specified as a
default fate of the A-line neural lineages. Whatever details are uncovered, it
is already clear that the distinct manner in which Nodal signals are involved
in neural patterning in vertebrates and ascidians implies a certain degree of
evolutionary plasticity in the mechanisms used to generate a conserved
structure such as the chordate neural tube.
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ACKNOWLEDGMENTS |
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