RESEARCH ARTICLE
285
Development 136, 285-293 (2009) doi:10.1242/dev.026419
Gene regulatory networks underlying the
compartmentalization of the Ciona central nervous system
Kaoru S. Imai1,2, Alberto Stolfi2, Michael Levine2 and Yutaka Satou1,*
The tripartite organization of the central nervous system (CNS) may be an ancient character of the bilaterians. However, the
elaboration of the more complex vertebrate brain depends on the midbrain-hindbrain boundary (MHB) organizer, which is absent
in invertebrates such as Drosophila. The Fgf8 signaling molecule expressed in the MHB organizer plays a key role in delineating
separate mesencephalon and metencephalon compartments in the vertebrate CNS. Here, we present evidence that an Fgf8
ortholog establishes sequential patterns of regulatory gene expression in the developing posterior sensory vesicle, and the
interleaved ‘neck’ region located between the sensory vesicle and visceral ganglion of the simple chordate Ciona intestinalis. The
detailed characterization of gene networks in the developing CNS led to new insights into the mechanisms by which Fgf8/17/18
patterns the chordate brain. The precise positioning of this Fgf signaling activity depends on an unusual AND/OR network motif
that regulates Snail, which encodes a threshold repressor of Fgf8 expression. Nodal is sufficient to activate low levels of the Snail
repressor within the neural plate, while the combination of Nodal and Neurogenin produces high levels of Snail in neighboring
domains of the CNS. The loss of Fgf8 patterning activity results in the transformation of hindbrain structures into an expanded
mesencephalon in both ascidians and vertebrates, suggesting that the primitive MHB-like activity predates the vertebrate CNS.
KEY WORDS: Ciona intestinalis, Gene regulatory network, Fgf8
1
Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku,
Kyoto, 606-8502, Japan. 2Department of Molecular and Cellular Biology, Division of
Genetics and Development, University of California, Berkeley, CA 94720, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 7 November 2008
property of the vertebrate CNS. The vertebrate MHB organizer
secretes Wnt1 and Fgf8, which are important for the MHB
organizing activity. Although the Ciona genome does not contain
Wnt1 (Hino et al., 2003), an ortholog of Fgf8 exhibits localized
expression during the development of the Ciona CNS at the late
gastrula stage (Imai et al., 2004; Hudson and Yasuo, 2005; Imai et
al., 2006) and at the middle tailbud stage (Imai et al., 2002), thereby
raising the possibility that an MHB organizer-like structure operates
in ascidians.
To determine the extent to which the compartmentalization of the
ascidian and vertebrate CNS are controlled by conserved and nonconserved gene circuits, we have examined the expression and
function of a comprehensive set of neural regulatory genes. These
studies permitted the reconstitution of a provisional gene regulatory
network for the development of the ascidian CNS based on
systematic gene disruption assays. The resultant network can explain
the causalities of the gene expression profiles seen in the CNS and
provide insights into the evolutionary origin of the vertebrate CNS.
MATERIALS AND METHODS
Ascidian embryos
C. intestinalis adults were obtained from the Maizuru Fisheries Research
Station of Kyoto University (Kyoto, Japan) and from Half Moon Bay harbor
(CA, USA). We used late gastrula embryos just after the neural plate
formation and late gastrula embryos just prior to the next division in the
present study, and early and mid-tailbud embryos, which correspond to E55
and E75-E80 (Cole and Meinertzhagen, 2004).
Expression profiles of regulatory genes
Most cDNA clones were obtained from our EST collection (Satou et al.,
2002). The detailed procedure for whole-mount in situ hybridization has
been described (Satou and Satoh, 1997). Cell identities were determined by
DAPI staining of nuclei.
Gene knockdowns
MO oligonucleotides were purchased from Gene Tools: BMP2/4,
AAGTCCAATCCGTAAGCGCCACCAT; Cdx, TTGTGCGTTTCTCATCAATGGTTGC; Delta-like, GAAGTAATATAAGCTTGATGCTCAT;
DEVELOPMENT
INTRODUCTION
The expression profiles of neural genes in both protostomes and
deuterostomes suggest that the tripartite structure of the CNS might
be evolutionarily ancient (Wada et al., 1998; Lowe et al., 2003;
Reichert, 2005). The vertebrate CNS has elaborated on this basic
tripartite structure to produce complexity and novelty. The anterior
neural tube develops into the brain, whereas posterior regions form
the spinal cord. The brain becomes regionalized into forebrain,
midbrain and hindbrain, which are further subdivided at later stages.
The MHB organizer is one of the mechanisms used for the
elaboration of the complex vertebrate brain (Liu and Joyner, 2001;
Wurst and Bally-Cuif, 2001; Rhinn et al., 2006). A number of
transcription factors and signaling molecules are expressed in the
MHB region, and these molecules constitute a complex genetic
network that establishes and maintains features of the organizer.
Fgf8 and Wnt1 are the key factors in this network controlling the
development of the midbrain and hindbrain.
The ascidian tadpole represents the closest living relative of the
vertebrates and possesses a simplified CNS derived from the dorsal
neural tube. The ascidian CNS consists of a centralized sensory
vesicle (SV), visceral ganglion (VG) and nerve cord composed of
~260 cells (Meinertzhagen et al., 2004). There is a morphologically
distinguishable domain called the ‘neck’ between the SV and VG
(Nicol and Meinertzhagen, 1988). Although the SV corresponds to
the forebrain, it is unclear whether the neck and VG correspond to
the hindbrain and/or spinal ganglia of vertebrates (Wada et al., 1998;
Dufour et al., 2006). It is conceivable that a distinct midbrain
counterpart is absent in the ascidian larva (Takahashi and Holland,
2004). Therefore, the MHB organizer has been regarded as a novel
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Development 136 (2)
Fig. 1. Development of the A- and b-line central nervous system in the Ciona embryo. (A) Schematic representations of A- and b-line neuronal
cell lineages of the bilaterally symmetrical embryos at the late gastrula stage. Cell names are shown in the lower panel. (B,C) A schematic representation
of the CNS cells at the tailbud stage from the (B) dorsal and (C) lateral views, and their lineages. Arrows indicate cell lineages and the cells with the same
colors are derived from the single cells at the late gastrula stage (enclosed by pink lines). Cells enclosed by thick black lines are post-mitotic cells destined
to become motoneurons. At the early tailbud stage, three pairs of presumptive motoneurons are post-mitotic. Until the mid-tailbud stage, two pairs of
post-mitotic presumptive motoneurons are differentiated from the remaining two pairs of the visceral ganglion (VG) cells after cell divisions, as shown in
the upper part of B. (D) The central nervous system of a tailbud embryo developed from an egg electroporated with Fgf8/17/18>RFP/AchTP>GFP. (E) A
late tailbud embryo developed from an egg electroporated with Fgf8/17/18>RFP and AchTP>GFP. Arrowheads indicate motoneurons. (F) The central
nervous system of a tailbud embryo developed from an egg electroporated with Fgf8/17/18>RFP / FoxB>GFP. RFP marks the A9.30-descendants and GFP
marks neurons. LNC, the lateral rows of the nerve cord; DNC, the dorsal row of the nerve cord; VNC, the ventral row of the nerve cord.
GTGGAGATTTCAAGTATGACAT) and Neurogenin (ATCGGTTTGCAGAATAATCCAACAT) of Ciona savignyi, a closely related species, into
Ciona savignyi eggs. We carried out in situ hybridization of Otx and
Pax2/5/8-A for Fgf8/17/18 morphant embryos, of Fgf8/17/18 for Snail
morphant embryos, of Snail for Neurogenin morphant C. savignyi embryos
and of Cyp26, FoxB and Hox1 for Otx morphant C. savignyi embryos (see
Fig. S1 in the supplementary material). They recapitulated the original
phenotypes. All of these observations support specificities of the MOs used
in the present study. Whole-mount in situ hybridization was used for
determining genes expressed in the downstream of genes that were
knocked down.
Electroporation
4.8 kb and 4.2 kb of flanking 5⬘ sequences of Fgf8/17/18 and FoxB
respectively were PCR-amplified from genomic DNA. AchTP promoter was
obtained as described previously (Yoshida et al., 2004). All were cloned into
pCESA vector upstream of unc-76-tagged GFP or mCherry reporter gene
(Dynes and Ngai, 1998). The detailed procedure for electroporation has been
described previously (Corbo et al., 1997).
RESULTS
The ascidian brain has distinct domains with
different properties
According to previous descriptions (Nicol and Meinertzhagen,
1988), the posterior region of the sensory vesicle (PSV) and VG are
derived from the posterior neural plate (Fig. 1A). The short region
DEVELOPMENT
Emc, CAACTTTAACCATTTTGCTGATTCT; en, TGGGCAACCTTGTATTTCGCTTCAT; ephrinA-b, ACAAAGGCATGGTGATATACGCATT; Fgf8/17/18, TACTCGCAATGCATTAAATCCGAAT; FoxB,
AGTCTCGTCCTGGTCGTGGCATTTT; Gli, CGCGTTCTCCATGTAAAATCTACGA;
Hedgehog2,
ACTGTCCCGCTTATACGTTACTCGC; Hnf6, CAGACTGACCGAGCGAAACTGGCAT; Irx-C,
TAAGACCGGGCAGCTCCGACTGCAT; Lhx3, AGAATTAACTGTAACAAATTGATCG; Lmx, TTTCGTCGTTAGAAGAACGCAGCAT; Mnx,
CTGACTTTGAAGTACTTAGCATCAT; Msxb, ATTCGTTTACTGTCATTTTTAATTT; Neurogenin, AAATCCAACATTTTGTAGCAAGAGC; Nk6, GAACCAGATTCTTCCATGGACATCA; Otx, CATGTTAGGAATTGAACCCGTGGTA;
Pax2/5/8-A,
CAGTTCATATTCAAACTTACTAACA; Pax3/7, AATTAGACCCTGGATGCATCATGTT;
Pax6, GCCTACAAGAATCGTACGTCGCCAT; Snail, GTCATGATGTAATCACAGTAATATA; SoxB1, AACATGAAGTCGTTCTGAGATGGCT; Tbx2/3, GAGGTCCACACCAACACTTTAACAT; and Raldh2,
GTACTGCTGATACGACTGAAGACAT.
Microinjections
were
performed as described (Imai et al., 2000). Blast searches of the MO
sequences could find no possible mis-targeting sites in the genome
sequence (Dehal et al., 2002), and we found no relations that cannot be
explained from the expression profiles. Different effective MOs produced
different phenotypes (see Fig. S1 in the supplementary material), and we
could not find any effects in embryos injected with an MO against lacZ
(data not shown), indicating that there were no generalized disruptions. For
further confirmation of specificities of the MOs, we injected second MOs
against Fgf8/17/18 (CATTTTCGTATGTAATCCAAGAGAA) and Snail
(TATTTCACAGTGAGAATTTTAATAT), and MOs against Otx (AGT-
Compartmentalization of the Ciona CNS
A
RESEARCH ARTICLE
B
Cdx
COE
E(spl)/hairy-b
Emc
ets/pointed2
FoxA-a
FoxB
Lmx
Mnx
msxb
MYTF
Neurogenin
Pax3/7
Pax6
Snail
SoxB1
ZF (C2H2)-33
ZicL
A9.13
A9.14
A9.15
A9.16
A9.29
A9.30
A9.32
b9.37
b9.38
BMP2/4
Delta-like
EphrinA-b
EphrinA-d
FGF8/17/18
FGF9/16/20
C
287
Fig. 2. Expression profiles of the regulatory
genes. (A) A- and b-line neural cells at the late
gastrula stage, (B) posterior sensory vesicle (PSV),
neck, VG and nerve cord at the early tailbud stage,
and (C) VG at the middle tailbud stage. Each
expression is shown by a red rectangle. (D) A
hierarchical clustering of the neural cells based on
the expression profiles of transcription factor genes
shown in B.
Arix
COE
Emc
en
ets/pointed2
FoxA-a
FoxB
FoxD-a/b
Gli
HNF6
Hox1
Hox5
Irx-C
islet
Lhx3
Lmx
macho-1
Mnx
MYTF
Neurogenin
NK6
Otx
Pax2/5/8-A
Pax3/7
Pax6
SoxB1
Tbx2/3
Unc4-A
D
A11.64
A11.63
A11.62
A11.61
A11.120
A11.119
A11.116
A11.115
A11.126-8
A11.118
A11.117
A10.57
b10.75
b10.76
b10.74
b10.73
A10.28
A10.27
A10.26
A10.30
A10.25
A10.29
Delta-like
EphrinA-d
FGF9/16/20
Hedgehog2
Wnt7
Chox10
COE
Emc
en
FoxB
HNF6
Hox1
Hox3
Irx-C
islet
Lhx1
Lhx3
Mnx
MYTF
Neurogenin
NK6
Pax6
POU4
SoxB1
PSV neck
LNC
posterior VG
anterior VG
DNC
VNC
A12.240
A12.238
A13.474
A13.473
A12.239
A11.118
A11.117
A10.57
Delta-like
EphrinA-b
EphrinA-d
FGF8/17/18
VG of older embryos (anterior two yellow cells, Fig. 1E). The
posterior-most pair of motoneurons (A10.57) arises from the A9.29
lineage (posterior green cells, Fig. 1D,E). Embryos electroporated
with a GFP fusion gene containing the FoxB enhancer permitted
visualization of the anterior lineage arising from A9.16, which forms
the PSV. As seen for endogenous FoxB transcripts, GFP is strongly
expressed in the anterior A9.16 lineage that forms the PSV, but not
in the posterior A9.16 lineage that forms the neck region (Fig. 1F).
The neck consists of quiescent undifferentiated precursors that form
the neurons homologous to cranial motoneurons of vertebrates after
metamorphosis (Dufour et al., 2006). These molecular data
confirmed the idea that the PSV, neck and VG have distinctive
properties (Cole and Meinertzhagen, 2004).
The regulatory states of cells in the developing
CNS
An earlier study identified a comprehensive list of regulatory genes
expressed in the developing nervous system (Imai et al., 2004). The
recent advances in imaging technology and the knowledge of the
cell lineages of the CNS (Cole and Meinertzhagen, 2004) allowed
us to elaborate on this description at single cell resolution by in situ
hybridization (summarized in Fig. 2A-C; see also in situ
DEVELOPMENT
between the PSV and VG is called the ‘neck’. Both the PSV and
neck are derived from a single pair of neuronal progenitor cells in
gastrulating embryos, A9.16, whereas the VG is derived from two
neighboring blastomeres, A9.30 and A9.29 (Fig. 1B). The latter cell
also contributes to the caudal nerve cord, which consists of nonneuronal ependymal cells. The ventral and dorsal rows of the neural
tube are derived from A9.13/A9.14/A9.15 and b9.37/b9.38,
respectively (Fig. 1C).
It is thought that the PSV and VG produce the core synaptic
activity for the swimming behavior of the tadpole, because the VG
includes five pairs of motoneurons that innervate the tail muscles
and are modulated by cholinergic neurons located in the PSV (Cole
and Meinertzhagen, 2004). To confirm this idea, we traced the A9.30
lineage with an RFP fusion gene containing the Fgf8/17/18 enhancer
(Fig. 1D-F) throughout development, as Fgf8/17/18 expression is
restricted to A9.30 at the late gastrula stage (Imai et al., 2004). Coelectroporation of this fusion gene with a GFP fusion gene
containing the enhancer of the acetylcholine transporter gene
(AchTP) identifies two pairs of early post-mitotic cholinergic
motoneurons derived from A9.30 (yellow cells, Fig. 1D). The
anterior A9.30 lineage forms non-neuronal ependymal cells (red
cells, Fig. 1E), as well as two additional pairs of motoneurons in the
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Development 136 (2)
Fig. 3. Gene regulatory networks
in the ascidian CNS. (A) Summary of
these networks. (B-E) Diagrams of
gene circuits in (B) A11.64 (PSV),
(C) A11.62 (neck), (D) A11.118 (VG)
and (E) A11.116 (caudal nerve cord) at
the early tailbud stage. The color code
for cells is the same as in Fig. 1.
Transcription factor genes and
signaling ligand genes are indicated by
rectangles and ovals, respectively.
Genes expressed in the ancestors of
the cell but not expressed at this stage
are enclosed by broken lines. Genes
that are not expressed in either the
specified cell or its ancestors are
shown in gray. Arrows indicate
transcriptionally regulatory
interactions. The lines ending in a bar
indicate repression. See the text for
details.
A
Arix
BMP2/4
Cdx
Chox10
COE
Delta-like
E(spl)/hairy-b
Emc
en
EphrinA-b
EphrinA-d
ets/pointed2
FGF8/17/18
FGF9/16/20
FoxA-a
FoxB
FoxD-a/b
Gli
Hedgehog2
HNF6
Hox1
Hox3
Hox5
Irx-C
islet
Lhx1
Lhx3
Lmx
macho-1
Mnx
msxb
MYTF
Neurogenin
NK6
Otx
Pax2/5/8-A
Pax3/7
Pax6
POU4
Snail
SoxB1
Tbx2/3
Unc4-A
Wnt7
B
C
D
E
A11.118
A11.62
A11.116
A11.64
E(spl)/hairy-b
Delta-like
COE
Delta-like
E(spl)/hairy-b
Emc
E(spl)/hairy-b
Emc
Emc
en
en
FGF8/17/18
EphrinA-d
FGF8/17/18
FGF9/16/20
FoxB
FGF9/16/20
FoxB
HNF6
Hox1
Hox1
MYTF
Gli
Hox1
Irx-C
Lhx3
Otx
Pax3/7
MYTF
Otx
Mnx
Neurogenin
Pax6
SoxB1
Pax2/5/8-A
Pax3/7
NK6
Pax6
Pax6
SoxB1
Snail
SoxB1
PSV neck
VG
Cdx
COE
Delta-like
E(spl)/hairy-b
en
EphrinA-b
EphrinA-d
FoxA-a
Hox1
Hox5
Lhx3
Mnx
MYTF
Neurogenin
Pax6
Snail
SoxB1
LNC
hybridizations of control embryos in Fig. S1 in the supplementary
material). The resultant diagrams of the expression profiles of
individual cells define the regulatory states of the cells from the late
gastrula to the mid-tailbud stage.
The hierarchical clustering of the expression profiles confirmed
and refined the morphological differences of the brain regions from
a molecular viewpoint (Fig. 2D). Two pairs of the PSV cells (A11.63
and A11.64 in the early tailbud embryo) are in the same regulatory
state, based on selective expression of Otx, FoxB and En. The neck
cells (A11.61 and A11.62) selectively express Pax2/5/8-A, Hox1 and
Gli. The VG and caudal nerve cord cells express different
combinations of regulatory genes. Anterior and posterior
motoneurons in the VG express Hox1/En/Neurogenin and
Lhx3/Hnf6/Neurogenin, respectively. Posterior portions of the neural
tube express Cdx, Snail and Pax6. The dorsal and ventral rows of the
neural tube also express different sets of regulatory genes.
Provisional gene regulatory networks in the
developing CNS
On the basis of the expression profile data, we systematically
perturbed the functions of regulatory genes expressed in the CNS
with specific morpholino oligonucleotides (MOs) in order to
determine the molecular basis for the compartmentalization of the
Ciona brain. We succeeded in disrupting the activities of a total of
25 regulatory genes (Tables 1 and 2). In situ hybridization assays
were used to monitor the effects of the different MO-induced
mutants on the expression of all of the regulatory genes expressed at
the stages examined (see Fig. S1 and Table S1 in the supplementary
material). This information, along with earlier results (Imai et al.,
2002; Lemaire et al., 2002; Hudson and Yasuo, 2005; Moret et al.,
2005; Imai et al., 2006; Hudson et al., 2007; Ikuta and Saiga, 2007),
was used to create a provisional circuit diagram showing the
interconnections among the regulatory genes controlling the
regionalization of the CNS (Fig. 3A). The simplicity of the system
permits the elucidation of gene networks at single cell resolution
(Fig. 3B-E; see Figs S2-S4 in the supplementary material).
For example, Fgf9/16/20 expression in the PSV and neck
progenitors is controlled by Emc and Pax3/7 (Fig. 3B). In the neck
region, Pax2/5/8-A also contributes to the expression of Fgf9/16/20
(Fig. 3C). Neurogenin was found to be a crucial determinant of the
motoneurons in the VG (Fig. 3D). Cdx is required for the development
of ependymal cells in posterior regions of the neural tube (Fig. 2E),
whereas Lmx contributes to specification of the dorsal nerve cord cells
by activating Pax3/7 (see Fig. S3 in the supplementary material).
DEVELOPMENT
Delta-like
Arix
Compartmentalization of the Ciona CNS
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289
Table 1. Summary of knockdown experiments with morpholino oligonucleotides for genes expressed at the late gastrula stage
Number of genes
A Genes analyzed in detail
Genes whose downstream genes were found
Cdx, Bmp2/4, Delta-like, Emc, Fgf8/17/18, FoxB, Lmx, Mnx, Msxb, Neurogenin, Pax3/7, Pax6, Snail, SoxB1
14
B Genes whose functions in the later development were hardly examined, because of their expression in the early embryo
ets/pointed2, EphrinA-d, FGF9/16/20, FoxA-a, ZicL
5
C Genes for which no good morpholino oligonucleotides were obtained
COE, E(spl)/hairy-b, Mytf, ZF-C2H2-33
Localized Fgf8/17/18 delineates PSV and neck
regions of the Ciona CNS
The reconstituted networks reveal a central role of Fgf8/17/18 in
generating regional patterns of gene expression. Fgf8/17/18 is
expressed at the late gastrula stage in A9.30 (see Fig. 5C), which
forms most of the VG (A11.117 through A11.120) at later stages.
This Fgf signal acts on the neighboring A9.16 cell to define the neck
region of the definitive tadpole CNS, which forms between the PSV
and VG. Otx and FoxB expression is normally restricted to the
anterior-most regions of the CNS, including the presumptive PSV
(Fig. 4A; see Fig. S1Q in the supplementary material; see also Fig.
2B). There is a posterior expansion of both expression patterns in
morphant embryos injected with an Fgf8/17/18 MO (Fig. 4B; see
Fig. S1Q in the supplementary material). Thus, Fgf8/17/18 inhibits
Otx and FoxB expression in the neck, thereby restricting their
activities to the PSV. En normally displays periodic expression in
the PSV and VG (Fig. 4C), but expression extends into the neck of
morphant embryos (Fig. 4D). There is also a loss of Pax2/5/8-A
expression (Fig. 4F), which is normally restricted to a tight stripe of
expressing cells in the neck (Fig. 4E). Gli, Fgf9/16/20 and Arix,
which are normally expressed in the neck, are lost in morphant
embryos, because these genes are under the control of Pax2/5/8-A
(see Fig. S1A,O,S in the supplementary material). Finally, Hox1
expression is normally restricted to the neck and VG (Fig. 4G), but
expression is lost in the neck of morphant embryos (Fig. 4H).
Altogether, the network analysis suggests that the neck is not formed
in Fgf8/17/18 morphants, but is transformed into an expanded PSV
(Fig. 4I,J).
Fgf8/17/18 is first expressed at the 64-cell stage in A7.6, which
abuts A7.8 (a progenitor of A9.29-A9.32, the VG and caudal nerve
cord lineage cells), but this expression disappears before the late
gastrula stage. Although this early expression is also suppressed in
4
the Fgf8/17/18 morphant, this early Fgf8/17/18 expression is not
likely to be required for the CNS regionalization, because Pax2/5/8A is not expressed in embryos treated with a MEK inhibitor, U0126,
from the late gastrula (Fig. 5). The same Fgf gene is again expressed
in the VG at the middle tailbud stage (Imai et al., 2002), but this
expression is later than Pax2/5/8-A expression. Therefore, the
expression in A9.30 at the late gastrula stage is most likely to be
responsible for the regionalization of the CNS.
This phenotype of Fgf8/17/18 morphants is evocative of the
transformation of the metencephalon into an expanded
mesencephalon seen in ace (Fgf8a) mutants in the zebrafish CNS
where there is expanded expression of Otx2 (mesencephalon) and
a loss of Pax8 (metencephalon) (Jaszai et al., 2003). Similar
transformations are also seen in conditional knockout mice of
Fgf8 (Chi et al., 2003). Thus, in the vertebrate CNS, Fgf8 is
expressed in the MHB region and required for proper
specification of the midbrain and anterior hindbrain. We might
therefore be able to regard the PSV and neck as counterparts of
the vertebrate midbrain and anterior hindbrain, although this has
been a debatable issue (Wada et al., 1998; Takahashi and Holland,
2004; Canestro et al., 2005; Dufour et al., 2006). Regardless of
their exact evolutionary counterparts and the timing of the
signaling, we propose that the recruitment of Fgf8 signaling might
have been a crucial event for the compartmentalization of the
chordate brain. It is less likely that the same signaling system was
independently acquired for similar uses by tunicates and
vertebrates.
Despite the apparent similarities in the patterning of the ascidian
PSV and neck with the compartmentalization of the vertebrate
midbrain and anterior hindbrain, we note a number of differences in
these processes. First, Ciona Fgf8/17/18 acts much earlier – during
late gastrulation – than it does in vertebrate embryos. Fgf8 might act
Table 2. Summary of knockdown experiments with morpholino oligonucleotides for genes expressed in the brain, boundary and
visceral ganglion at the early tailbud stage
Number of genes
A Genes analyzed in detail
Genes whose downstream genes were found
Emc, FoxB, Delta-like, EphrinA-b, Gli, Hnf6, Lmx, Mnx, Neurogenin, Otx, Pax2/5/8-A, Pax3/7, Pax6, SoxB1
14
En, Hedgehog2, Irx-C, Lhx3, Nk6, Tbx2/3
6
B Genes whose functions in the later development were hardly examined, because of their expression in the early embryo
EphrinA-d, ets/pointed2, Fgf9/16/20, FoxA-a, macho-1
5
C Genes not analyzed
Genes for which no good morpholino oligonucleotides were obtained
COE, Hox1, Mytf, Unc4-A, Wnt7
5
Genes staring to be expressed late in the early tailbud stage
Arix, FoxD-a/b, Hox5, islet
4
DEVELOPMENT
Genes whose downstream genes were not found
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RESEARCH ARTICLE
Development 136 (2)
Fig. 5. The Fgf signaling between the late gastrula stage and the
neurula stage is required for Pax2/5/8-A expression.
(A-D) Expression of Fgf8/17/18 from the 44-cell stage to the neurula
stage. Expression in A7.6 is shown by black arrowheads and expression
in the A9.30 is shown by white arrowheads. (E-H) Expression of
Pax2/5/8-A following U0126 treatment at different time points.
Numbers indicate the total number of embryos analyzed. The
developmental time point when embryos were placed in sea water
containing U0126 is shown in the top. Embryos treated with U0126
from the late gastrula stage when Fgf8/17/18 expression in A9.30
begins do not express Pax2/5/8-A, whereas embryos treated with
U0126 from the neurula stage express Pax2/5/8-A.
Fig. 4. FgF8/17/18 delineates PSV and neck regions.
(A-H) Expression of (A,B) Otx, (C,D) En, (E,F) Pax2/5/8-A and (G,H) Hox1
in A,C,E,G control embryos and (B,D,F,H) experimental embryos
developed from eggs injected with Fgf8/17/18 MO. Cell identities were
determined by DAPI staining of nuclei (inserts in A-H). Cells from the
posterior end of the SV (A11.63) to the middle part of the VG (A11.118
or its descendants) are enclosed by red and light-blue lines, which show
the expression or lack of expression of the indicated genes, respectively.
Broken white lines indicate the boundaries of the PSV/neck and the
neck/VG. Note that A11.119 is about to divide, or has recently divided,
into two daughter cells in A,B,E,F. For simplicity, they are enclosed by
single lines. The embryos shown in G and H are at a slightly later stage,
when Hox1 gene expression is more prominent. (I,J) Schematic
representations of the brain regionalization mechanism by Fgf8/17/18
in (I) the normal embryo and (J) Fgf8/17/18-morphant embryo. Note
that Fgf8/17/18 is not expressed in the VG at the tailbud stage but is
expressed in the progenitor cells (A9.30) in the neural plate.
early in the ancestral chordates, as seen in the Ciona embryos, or the
timing of Fgf8 signaling might have shifted to earlier stages during
the retrograde evolution of tunicates. Second, Wnt1 is absent in the
Localized expression of Fgf8/17/18 depends on the
Snail repressor
In vertebrate embryos, localized expression of Fgf8 in the MHB is
directed by the interaction of Otx and Gbx. However, restricted
expression of Fgf8/17/18 in the A9.30 lineage of ascidian embryos
does not depend on Otx and Gbx. MO-induced suppression of Otx
gene activity did not affect Fgf8/17/18 expression (see Fig. S1N in
the supplementary material). Moreover, Gbx is not present in the
Ciona genome (Dehal et al., 2002; Wada et al., 2003). In contrast to
vertebrates, Fgf8/17/18 is positively regulated by Nodal signals
emanating from the b-line neural cells (blue cells in Fig. 1) (Imai et
al., 2006). The key component of Fgf8/17/18 regulation is the
differential expression of the Snail repressor in the A9.30 and A9.32
neural progenitors. Snail expression is explicitly stronger in A9.32
than A9.30 (Fig. 6A). This augmented expression depends on
Neurogenin, which is expressed only in A9.32. MO-induced
suppression of Neurogenin resulted in reduced levels of Snail in
A9.32, similar to the levels normally seen in A9.30 (white
arrowhead, Fig. 6A⬘). This reduction in Snail causes ectopic
activation of Fgf8/17/18 in the A9.32 lineage (white arrowhead, Fig.
6B⬘). There is a similar de-repression of Fgf8/17/18 expression upon
MO-mediated disruption of Snail activity (Fig. 6C).
Neurogenin is under the control of Nodal signaling, and
suppression of Nodal via MO injection led to the complete
elimination of Snail expression in both A9.30 and A9.32 (Imai et
al., 2006). Thus, Nodal is sufficient to induce low levels of Snail
DEVELOPMENT
Ciona genome (Hino et al., 2003), and, hence, it is not involved in
the regionalization of the Ciona CNS. It is possible that the ancestral
chordate had a regionalization mechanism directed by Fgf8 and
Wnt1, but that Wnt1 was subsequently lost by ascidians. However,
in amphioxus, Wnt1 is not expressed around the boundary of the
putative midbrain and hindbrain, and therefore the ancestral
chordate might have relied solely on an Fgf8-dependent mechanism
for regionalization of the CNS.
Fig. 6. Snail directs localized expression of Fgf8/17/18.
(A-C) Expression of (A,A⬘) Snail and (B,B⬘,C) Fgf8/17/18 was examined
in (A,B) control unperturbed embryos, (A⬘,B⬘) Neurogenin-knockdown
embryos and (C) Snail-knockdown embryos. Red and white arrowheads
indicate A9.30 and A9.32, respectively. (Insets in A and A⬘) Highmagnification images of the left side of the embryos shown in A and
A⬘. A9.30 and A9.32 are enclosed by red and white lines, respectively.
Note that the embryos shown in B,B⬘ and C are slightly older than
those in A and A⬘. (D) A schematic diagram of regulation of Fgf8/17/18.
See the text for details.
expression in A9.30. These low levels fail to block Fgf8/17/18
expression, but might restrict the levels of expression. The
enhanced expression of Snail seen in A9.32 arises from a feedforward loop: Nodal induces Neurogenin and the two regulators
work together to activate Snail (Fig. 6D). These augmented levels
of Snail result in the complete repression of Fgf8/17/18 in the
A9.32 lineage of the CNS.
The localized expression of Neurogenin is achieved by two
signaling molecules. Nodal is expressed in the b-line cells (blue
cells in Fig. 1 and Fig. 6D), which are juxtaposed to A9.32 but not
A9.30. Similarly, Delta-like is expressed in the b-line cells under
the control of Nodal signaling (Hudson and Yasuo, 2005; Imai et
al., 2006), and the Delta-Notch signaling contributes to activation
of Neurogenin (see Fig. S1Ad in the supplementary material).
Thus, these two inputs induce localized expression of Neurogenin
in A9.32 cooperatively or by a simple cascade mechanism.
It has been proposed that the ancestral chordates had the
Otx/Gbx system but lacked an apparent MHB organizer, as the
Otx and Gbx expression patterns abut in amphioxus CNS,
although other components of the MHB organizer such as En,
Pax2/5/8 and Wnt1 are not expressed at this boundary (Castro et
al., 2006). If so, the recruitment of Fgf8 signaling as an MHB
organizer might have evolved after the divergence of the
cephalochordates and tunicate/vertebrate lineages. There are two
possible scenarios for the advent of Fgf8 in the
compartmentalization of the vertebrate CNS. First, the Otx/Gbx
gene circuit might have been used to regulate Fgf8 in the ancestral
RESEARCH ARTICLE
291
Fig. 7. Retinoic acid is required for Hox1 expression. (A) Radlh2 is
expressed in the most anterior muscle cells at the tailbud stage (upper
panel, a lateral view; lower panel, a dorsal view). (B) Hox1 is expressed
in the neck region, the anterior part of the VG and the anterior part of
the caudal nerve cord within the CNS of control unperturbed embryos.
Red and yellow arrowheads indicate boundaries between the neck and
the visceral ganglion and between the visceral ganglion and the caudal
nerve cord, respectively. (C) Hox1 expression is not observed in RaldH1knockdown embryos. (D) A schematic diagram of regulation of Hox1.
FoxB represses Hox1, and this repression may be through Cyp26, which
is expressed under the control of FoxB. See the text for details.
chordate, and the switch in Fgf8 regulation to the
Nodal/Neurogenin/Snail gene circuit might reflect the streamlined
cell lineages in the early Ciona embryo and the need for the
precise regulation of Fgf8 at single-cell resolution. Alternatively,
Fgf8 regulation by the Nodal/Neurogenin/Snail gene circuit might
be ancient, and in vertebrates the upstream regulatory mechanism
was integrated into a pre-existing Otx/Gbx gene circuit. In this
regard, we note that orthologs of Fgf8 are regulated by Snail
during gastrulation of the Drosophila embryo (e.g. Stathopoulos
et al., 2004).
Hox1 expression is controlled by Fgf8/17/18
through retinoic acid signaling
As discussed earlier, Fgf8/17/18 signaling inhibits Otx expression
in the posterior A9.16 lineage, thereby restricting its expression
to the anterior lineage, the future PSV. Otx either directly or
indirectly activates the forkhead regulatory gene FoxB, which
represses Hox1 expression in the PSV. It is well known that Hox
genes are regulated by retinoic acid (RA) signaling in chordates
(Holland and Holland, 1996; Maden, 2002). It has previously
been shown that RA enhances Hox1 expression in the Ciona
embryo (Nagatomo and Fujiwara, 2003). RA is synthesized by
Raldh2 (retinaldehyde dehydrogenase 2), which is expressed in
the most anterior muscle cells at the tailbud stage (Fig. 7A)
(Nagatomo and Fujiwara, 2003). Indeed, knockdown of Raldh2
eliminates Hox1 expression (Fig. 7B,C). Endogenously
synthesized RA is therefore responsible for Hox1 expression.
Interestingly, Cyp26 is expressed in the presumptive PSV region
of the Ciona CNS, and this expression is lost in morphant
embryos injected with either Otx or FoxB MOs (see Fig. S1F in
the supplementary material). Cyp26 encodes an enzyme
responsible for degrading RA. These observations raise the
DEVELOPMENT
Compartmentalization of the Ciona CNS
RESEARCH ARTICLE
possibility that FoxB indirectly represses Hox1 expression in the
presumptive PSV by activating Cyp26, which in turn inhibits RA
signaling (Fig. 7D). Previous studies suggested a possible
connection between Fgf signaling and Hox expression (Irving and
Mason, 2000; Shimizu et al., 2006; Skromne et al., 2007) and
between Cyp26 and Hox expression in the vertebrate CNS
(Hernandez et al., 2007). We propose that this connection might
reflect a direct regulatory connection between the MHB organizer
and RA signaling.
DISCUSSION
The comprehensive analysis of CNS regulatory genes led to the
elucidation of a provisional gene network in the early Ciona tadpole.
These networks provide a number of key insights into the
compartmentalization of the chordate CNS. First, a localized Fgf8
signaling center was probably used by the last shared ancestor of
ascidians and vertebrates to delineate two regions of the chordate
brain (mesencephalon and metencephalon). Second, Fgf8 signaling
in Ciona leads to restricted expression of Otx and FoxB in the PSV,
as well as restricted expression of Pax2/5/8-A in the neck. Otx and
FoxB might inhibit Hox1 expression in the forebrain via Cyp26,
whereas Pax2/5/8-A might coordinate the expression of the
regulatory genes required for the differentiation of metencephalon
motoneurons, such as Phox2a/Arix (e.g. Engle, 2006). Finally,
although the regulatory genes responsible for the
compartmentalization of the vertebrate CNS (e.g. Otx, Pax2,
Neurogenin, etc.) exhibit comparable patterns of expression in the
Ciona CNS, there are both conserved and distinctive features of the
underlying mechanism. Localized Fgf8 signaling is used to deploy
these expression patterns in both systems, even though different
regulatory mechanisms are used to restrict Fgf8.
This research was mainly supported by Grants-in-Aid from MEXT (17687022)
to Y.S. and the NSF (IOB 0445470) to M.L. and partly by a grant-in-aid of the
gCOE program from MEXT to K.S.I. We thank Prof. Nori Satoh for the
generous use of his laboratory facilities.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/285/DC1
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Compartmentalization of the Ciona CNS