DEVELOPMENTAL DYNAMICS 233:1177–1193, 2005

REVIEWS–A PEER REVIEWED FORUM

Specification of Embryonic Axis and Mosaic

Development in Ascidians
Hiroki Nishida*

Setting up future body axes is the first important event before and at the beginning of embryogenesis. The
ascidian embryo is a classic model that has been used to gain insight into developmental processes for over
a century. This review summarizes advances made in this decade in our understanding of the
developmental processes involved in the specification of the embryonic axes and cell fates during early
ascidian embryogenesis. Maternal factors, including mRNAs, are translocated to specific regions of the egg
by cytoplasmic and cortical reorganization, so-called ooplasmic segregation, and specify the animal–vegetal
axis and the one perpendicular to it, which is defined as the anteroposterior axis in ascidians. Some
postplasmic/PEM RNAs that are anchored to cortical endoplasmic reticulum are brought to the future
posterior pole of fertilized eggs, and play crucial roles in posterior development. Following specification of
the animal–vegetal axis, nuclear localization of ␤-catenin takes place in the vegetal blastomeres; this
occurrence is important for the acquisition of the vegetal character of the blastomeres in later development.
Positioning of these maternal factors lead to subsequent cell interactions and zygotic gene expression
responsible for axis establishment and for cell fate specification. We describe how endoderm blastomeres in
the vegetal pole region emanate inductive signals mainly attributable to fibroblast growth factor. Marginal
blastomeres next to endoderm blastomeres respond differently in ways that are determined by intrinsic
competence factors. Expression patterns of developmentally important genes, including key transcription
factors of each tissue type, are also summarized. Developmental Dynamics 233:1177–1193, 2005.
© 2005 Wiley-Liss, Inc.

Key words: embryonic axis; cell fate specification; ascidian; maternal factor; localized mRNA; embryonic induction; cell
signaling; transcription factor

Received 4 January 2005; Revised 14 March 2005; Accepted 28 March 2005

INTRODUCTION ascidian embryo is a classic model 2001; Nishida, 2002a,b; Satoh,

The three-dimensional structure of or-
ganisms is characterized by the three
perpendicular body axis, namely, the
anterior–posterior, dorsal–ventral, and
left–right axes. These axes generally
emerge in the early embryonic stage,
and are sometimes already apparent
in eggs before fertilization. One of the
major issues in developmental biology
is to understand how axial polarity is
achieved in a developing embryo. The
that has been used to study axis and
cell fate specification during embryo-
genesis for over a century (Chabry,
1887; Conklin, 1905; reviewed by Sa-
toh, 1994; Nishida, 1997; Jeffery,
2001). Recent advances in ascidian re-
search have placed this simple organ-
ism in a unique position to contribute
to a detailed understanding of the cel-
lular and molecular processes of em-
bryogenesis (reviewed by Corbo et al.,
2003a,b). Most of the recent concepts
and analysis of ascidian development
derive from observations and experi-
mental works using four main evolu-
tionarily distant species (Ciona intes-
tinalis and savignyi, Halocynthia
roretzi, and Phallusia mammillata),
which have contributed complemen-
tary and/or converging or diverging
information.
The restriction of cell fate proceeds

Department of Biology, Graduate School of Science, Osaka University, Osaka, Japan

Grant sponsors: MEXT; JSPS; Toray Science Foundation.
*Correspondence to: Hiroki Nishida, Department of Biology, Graduate School of Science, Osaka University, 1-1 Machi-
kaneyama-cho, Toyonaka, Osaka 560-0043, Japan. E-mail: hnishida@bio.sci.osaka-u.ac.jp
DOI 10.1002/dvdy.20469
Published online 22 June 2005 in Wiley InterScience (www.interscience.wiley.com).

© 2005 Wiley-Liss, Inc.

1178 NISHIDA
quickly in ascidian embryos. Just be-
fore gastrulation is initiated at the
110-cell stage, the developmental
fates of most blastomeres are re-
stricted such that a blastomere gives
rise to a single cell type in the tadpole
larva (Fig. 1; Nishida, 1987). Even
when blastomeres are isolated from
110-cell embryos, they continue to de-
velop into differentiated cells accord-
ing to the fate map (Reverberi and
Minganti, 1946; Nishida, 1992a). This
intriguing cell-autonomous property
of ascidian embryos is referred to in
many textbooks as a typical example
of mosaic development. Therefore, it
will be sufficient to take into account
the events that happen before the 110-
cell stage to fully understand axis
specification in this animal. This arti-
cle summarizes the current knowl-
edge on maternal and zygotic pro-
cesses related to embryonic axis
specification in ascidians. In this arti-
cle, we first describe postfertilization
processes involving maternal factors
before zygotic gene expression is initi-
ated. Then processes that require zy-
gotic gene expression and cellular in-
teractions after embryos become
multicellular will be summarized.
DEFINITION OF AXES IN
EARLY EMBRYOS
The definition of embryonic axis in
early embryos is somewhat artificial
and is a question of semantics. It is
difficult per se to superpose future em-
bryonic axes on eggs and embryos be-
fore and during gastrulation, because
dynamic movements and rearrange-
ments of cells occur in gastrulae. Dif-
ferences in axis definition between
amphibians and ascidians (which
show similar morphogenetic move-
ment) provide an example of the diffi-
culties (Fig. 2). In ascidians, the ani-
mal–vegetal (A-V) axis conventionally
has been considered to be the future
ventral– dorsal (V-D) axis, and the
axis perpendicular to the A-V axis is
the anterior–posterior (A-P) axis
(Conklin, 1905). By contrast, in Xeno-
pus, the definition of embryonic axis is
the opposite. The A-V axis is assigned
as the future A-P axis, and the D-V
axis is perpendicular to it (Nieuwk-
oop, 1977). The difference comes from
the difference in the position of the
developing embryo used as a reference
point (Arendt and Nübler-Jung,
1997). In this sense, the definition of
axes in early embryos is somewhat ar-
tificial. In studies of ascidians, the an-
imal pole, indicated by the site of polar
body extrusion, is always held up-
ward. As axial structures such as no-
tochord and nerve cord undergo con-
vergent and extension movement, and
thus the dorsal blastopore lip of ascid-
ian gastrulae heads vegetally, the
original vegetal pole is covered by
these dorsal tissues. Thus, the vegetal
pole corresponds to the future dorsal
side in ascidians. Looked at this way,
embryos lie in a somewhat unusual
way on their back. This finding may
not matter to ascidian researchers, be-
cause ascidian embryos develop in any
orientation in seawater like sea ur-
chin embryos. In contrast, in a usual
textbook drawing of amphibian gas-
trulation (e.g., Gilbert, 2003), the dor-
sal blastopore lip is held in a constant
position. In this presentation, one
holds the region of dynamic cell move-
ment and actually holds no cell
constantly. With this as a fixed point,
it is necessary to turn other regions
around such as the regions with
less dynamic cell movement, for exam-
ple, animal pole cells. This presenta-
tion makes sense in light of the fact
that amphibian embryos settle and
are oriented by gravity. When one
draws gastrulating embryos of either
animal in another way, one can see
how morphogenetic movements are
conserved between ascidians and am-
phibians.
In ascidians, the A-P axis of the
early embryo (corresponding to the
D-V axis in amphibians) is defined as
perpendicular to the A-V axis, as the
animal pole represents the reference
point. But it does not correspond pre-
cisely to the A-P axis of larvae, be-
cause cells do not remain stationary
during morphogenetic movements.
Complicated positional rearrange-
ment does not take place in the animal
hemisphere, and animal cells just
cover the embryo to form ectoderm by
epiboly, so the A-P axis of eggs and
early embryos in the animal hemi-
sphere corresponds precisely to the
A-P axis of larvae. Anterior-animal
blastomeres give rise to head/trunk
epidermis of larvae, and posterior-an-
imal blastomeres develop into tail epi-
dermis. The polar bodies attach to the
ventral midline at the boundary of
trunk and tail of the tadpole (Fig. 2).
What happens in the vegetal hemi-
sphere is not so simple (Fig. 1). Ante-
rior-vegetal blastomeres give rise
mainly to endoderm, notochord, and
nerve cord. Notochord and nerve cord
cells are located in the larval tail. Pos-
terior-vegetal blastomeres develop
mainly into endoderm, mesenchyme,
and muscle. Muscle cells are found in
the tail, while endoderm and mesen-
chyme cells are found in the trunk
region. In the present paper, “ante-
rior” and “posterior” are used in the
way these adjectives are traditionally
applied to ascidian eggs and early em-
bryos. Thus, in the present context,
“anterior fate” is that of blastomeres
of the anterior half of cleavage-stage
embryos and does not mean the fate
that gives rise to the anterior trunk
structures of larvae, nor does “poste-
rior fate” mean the fate that gives rise
to the posterior tail structures.
POLARITY IN
UNFERTILIZED EGGS
Generally, the A-V axis is already
present in unfertilized eggs. The axis
in pregastrula embryos is correlated
with germ layer segregation. In as-
cidians, ectoderm, mesoderm, and
endoderm territory is present along
the A-V axis, as in amphibian (Fig. 1).
Processes that involve maternally
stored factors and occur before the ac-
tivation of the zygotic genome are dis-
cussed in this and the following two
chapters. The mature oocytes of most
animals show radial symmetry with a
single A-V axis. The animal pole is
where polar bodies form during meio-
sis. Unfertilized eggs of ascidians are
in meiotic metaphase I with the mei-
otic spindle at the animal pole. In ad-
dition to the spindle, ascidian eggs
already show various kinds of polar-
ization along the A-V axis in the sub-
cortical and cortical domains (Fig. 3).
No remarkable polarization in the
central egg cytoplasm has been re-
ported. In the subcortical region, mi-
tochondria are enriched in the vegetal
half forming a mitochondria-rich re-
gion, called the myoplasm, from which
microtubules are excluded (Zalokar
and Sardet, 1984). The localization of
mitochondria in myoplasm would
have an adaptive significance in that
 AXIS SPECIFICATION IN ASCIDIANS 1179

Fig. 1. Developmental fates of blastomeres of the ascidian embryo.

A: Fate restriction during cleavage stages. Blastomeres are colored
when the fate of the blastomere is restricted to give rise to a single kind
of tissue cell. The colors correspond to the colors of larval tissues
indicated in C. Fate restriction in ascidian embryos proceeds quickly in
the early embryo. Sister blastomeres of the previous cleavage are con-
nected by bars. B: Fate map of the 110-cell stage. Animal and vegetal
hemispheres. Names of blastomeres are indicated as “a8.19” and so on.
C: Organization of tail bud embryos. Mid-sagittal planes, sagittal planes,
and transverse sections of the tail. TCL, trunk lateral cell; TVC, trunk
ventral cell.

they are preferentially segregated into

larval muscle cells, which need vigorous
energy production. In the egg cortex,
microfilaments form a basket with its
opening at the animal pole region (Sar-
det et al., 1992). The microfilaments
gives a mechanical motive force for the
first phase of cytoplasmic and cortical
reorganization, called ooplasmic segre- Fig. 2. Schematic representation of morphogenetic movements during
gation, as mentioned two paragraphs gastrulation of ascidians (left) and frogs (right). Presumptive notochord
region is colored pink. Anterior and posterior neural tube is colored light
below. A network of tubules and sheets and dark blue. Polar bodies indicate the animal pole. Green asterisks
of cortical endoplasmic reticulum (cER) point the original vegetal pole. The red arrowhead represents the point
is present just beneath the plasma held in a constant position. Embryonic axes at tail bud stage are indi-
membrane, showing a gradient with the cated at the bottom. See text in detail.
highest density at the vegetal pole. The
cER is tethered to the plasma mem- ternal mRNAs, called type I postplas- 1996), show a polarized distribution
brane (Sardet et al., 1992). mic RNAs in Halocynthia (Sasakura with the highest concentration at the
In addition to these membranous and et al., 2000) or PEM (posterior end vegetal pole cortex (Fig. 3) (reviewed
cytoskeletal components, several ma- mark) RNAs in Ciona (Yoshida et al., in Nishida, 2002a; Sardet et al., 2005).
1180 NISHIDA

Fig. 3. Localization of maternal factors from fertilization to the 110-cell stage. Cytoplasmic and cortical reorganization (ooplasmic segregation)
proceeds in two major phases during the first cell cycle. The centrosome-attracting body (CAB) is present in the posterior region of the B4.1
blastomeres of the eight-cell embryos and is inherited by the posterior-most blastomeres during cleavages. Cortical endoplasmic reticulum (cER) and
postplasmic/PEM RNAs are concentrated into the CAB. The bottom part shows localization of various maternal determinants as inferred from ooplasmic
removal and transplantation experiments. Nuclear localization of ␤-catenin during cleavages is shown at the bottom right.

These type I postplasmic/PEM RNAs tion in the unfertilized egg cortex, but clearly involved in the formation of
encode a great variety of protein their role in the establishment of the the A-P axis, as their final destination
types. They show a polarized distribu- A-V axis is unclear. Some of them are after ooplasmic movements is the pos-

terior region, as discussed in the next
section. The results of transplantation
of the peripheral cytoplasm have also
revealed the polarized distribution of
tissue-forming substances in unfertil-
ized eggs. Muscle and endoderm form-
ing activities are present in a gradient
manner with the highest activity at
the vegetal pole, while epidermis
forming activity is present in an oppo-
site gradient (Nishida, 1992b, 1997,
and references therein; Fig. 3). Thus,
unfertilized eggs already show appar-
ent polarity; however, when and how
the polarity is established during oo-
genesis and oocyte maturation is un-
known. In Halocynthia oocytes, the
germinal vesicle is in an eccentric po-
sition (Numakunai, 2001), but this
finding does not seem to be the case in
Ciona and Phallusia oocytes.
POSTFERTILIZATION
PROCESSES THAT
ESTABLISH THE ANIMAL
VS. VEGETAL CELL FATES
Cytoplasmic and Cortical
Reorganization During the
First Cell Cycle
Eggs of many kinds of animal show
cortical and cytoplasmic movements
just after fertilization. These pro-
cesses have been extensively analyzed
in Caenorhabditis elegans, Xenopus,
and ascidians. The mechanisms in-
volved and factors translocated are di-
verged among these animals; how-
ever, egg cortices commonly play a
significant role. The cortex provides a
scaffold to anchor and translocate lo-
calized maternal factors, and supplies
spatial information for later embryo-
genesis (reviewed by Sardet et al.,
1994, 2002). Ascidian eggs undergo
dramatic cytoplasmic and cortical re-
organizations between fertilization
and the beginning of the first cleav-
age; this process has been called oop-
lasmic segregation (Fig. 3; Conklin,
1905; Sardet at al., 1989; Roegiers et
al., 1999). Reorganizations occurs in
two major phases. Shortly after fertil-
ization (0 –10 min after insemination
in Halocynthia, 0 –5 min in Ciona and
Phallusia), the first phase of ooplas-
mic segregation is accompanied by a
rapid contraction of the egg cortex and
plasma membrane, resulting in a seg-
regation of microfilaments, mitochon-
AXIS SPECIFICATION IN ASCIDIANS 1181
dria, cER, postplasmic/PEM RNAs,
and muscle- and endoderm-forming
activity toward the vegetal pole region
(Roegiers et al., 1999; Nishida, 1997;
Sardet et al., 2003). A microfilament
basket with its opening at the animal
pole contracts toward the vegetal pole,
bringing these components toward the
vegetal pole region. Eggs still show
radial symmetry along the A-V axis at
this stage, but the original polarity is
much intensified, as all components
mentioned above are highly concen-
trated at the vegetal pole region (Fig.
3). In Halocynthia, the contraction
pole is at the opposite position of the
polar bodies in most cases. However,
in Phallusia and Ciona, the contrac-
tion pole can be off the opposite posi-
tion by as much as 45– 60 degrees,
depending on the site of the sperm
entry (Roegiers et al., 1995), making it
difficult to define the A-V axis unam-
biguously.
The second phase of reorganization
(85–110 min in Halocynthia, 25– 45
min in Ciona and Phallusia) brings
most of these components toward the
future posterior pole (Fig. 3; Sawada,
1988; Sardet et al., 1989; Nishida,
1997; Roegiers et al., 1999) and is rel-
evant to specification of the A-P axis.
Exceptionally, factors involved in
endoderm formation and gastrulation
do not move toward the future poste-
rior pole and just expand their distri-
bution to the entire vegetal hemi-
sphere during the second phase (Fig.
3; Bates and Jeffery, 1987; Nishida,
1996). The significance of this second
phase in the A-P axis specification is
discussed in the next chapter.
␤-Catenin and Vegetal Cell
Fate Specification
Recently, molecular aspects of postfer-
tilization processes that is involved in
establishment of the A-V axis in sea
urchin embryos have been investi-
gated intensively. Dishevelled (Dsh),
glycogen synthetase kinase 3 (GSK-3),
and ␤-catenin are involved. These
molecules are signaling molecules in
the canonical Wnt pathway. The Wnt
signaling pathway is generally used in
cellular communication. However,
early embryos use only a downstream
part of the intracellular signal trans-
duction cascade in maternal mecha-
nisms that specify embryonic axes. In
amphibian and fish embryos, this ma-
ternal system specifies the D-V axis,
which is perpendicular to the A-V axis
(Miller and Moon, 1996; Heasman,
1997; Schneider et al., 1996; Miller et
al., 1999). However, the same path-
way is used to establish the A-V axis
in echinoderms (Wikramanayake et
al., 1998; Emily-Fenouil et al., 1998;
Logan et al., 1999; Weitzel et al.,
2003). The role of this pathway in
germ layer segregation would be con-
served among ancient metazoans, as
Cnidaria embryos also shows nuclear
␤-catenin in cells that invaginate
(Wikramanayake et al., 2003). In the
canonical Wnt signaling pathway,
Wnt proteins bind to the transmem-
brane receptor Frizzled and then acti-
vate Dsh, which in turn inhibits
GSK-3 activity. GSK-3 destabilizes
␤-catenin protein. Hence, inhibition of
GSK-3 activity results in ␤-catenin
stabilization and its translocation into
nuclei. ␤-catenin forms a complex
with the transcription factor Tcf to ac-
tivate specific gene expression (Cadi-
gan and Nusse, 1997). In the sea ur-
chin, nuclear accumulation of ␤-catenin
occurs in the vegetal region of cleaving
embryos, and this occurrence is a cru-
cial event in endomesoderm (vegetal
cell fates) specification. Dsh is re-
quired for the stabilization of ␤-cate-
nin in vegetal cells. Green fluorescent
protein–tagged Dsh protein is tar-
geted to the vegetal cortex of the un-
fertilized egg (Weitzel et al., 2003).
Therefore, Dsh seems a key localized
protein, although the authors argue
the possibility that Dsh activator is
also localized in the vegetal cortical
region, because overexpressed Dsh
mRNA cannot promote vegetal fate in
animal blastomeres.
In ascidians, Imai et al. (2000) re-
ported a role for ␤-catenin in vegetal
fate specification in Ciona intestinalis
and C. savignyi embryos. In this re-
gard, ascidian embryos show a simi-
larity to echinoderm embryos. Prefer-
ential ␤-catenin nuclear localization
occurred in the vegetal hemisphere in
cleavage-stage embryos (Fig. 3). When
mRNA encoding the stabilized form of
␤-catenin (lacking the phosphoryla-
tion sites for GSK-3) was injected into
eggs, nuclear ␤-catenin was also ob-
served in the animal hemisphere. In
these embryos, most embryonic cells
produced alkaline phosphatase, an
1182 NISHIDA
endoderm differentiation marker,
and the expression of an epidermis
marker, Cs-Epi1, was down-regu-
lated. The only exception concerned
primary muscle precursor cells, whose
fate was not altered by the overpro-
duction of ␤-catenin. To inhibit ␤-cate-
nin function in nuclei, ␤-catenin was
sequestered into a cell adhesion com-
plex by overproduction of cadherin. In
these embryos, nuclear staining with
␤-catenin antibody was abolished in
the entire embryo. Alkaline phospha-
tase expression was lost, and Cs-Epi1
expression expanded into the vegetal
hemisphere. The embryos did not gas-
trulate. The development of muscle
cells was not perturbed in these cad-
herin-overproducing embryos. Nei-
ther ␤-catenin mRNA nor protein was
localized to the vegetal cytoplasmic re-
gion of eggs and initial cleavage stage
embryos. This finding suggests that
the localized endoderm determinants
may be molecules that stabilize
␤-catenin in the vegetal hemisphere.
A promising candidate is Dsh, as has
been shown recently in sea urchins.
Identification of maternal endoderm
determinants and gastrulation deter-
minants in ascidians remains elusive
for the moment.
These observations indicate that
A-V axis establishment is mediated by
␤-catenin signaling. Endoderm (vege-
tal) fate requires ␤-catenin function,
whereas epidermis (animal) fate is
promoted by suppressing ␤-catenin
function. It is not known whether me-
soderm cells in the marginal zone of
the vegetal hemisphere, such as noto-
chord and mesenchyme precursors,
require intermediate stabilization of
␤-catenin within the cells or not. In
sea urchin, there is a gradient in
␤-catenin stability along the A-V axis
during early cleavage, and this gradi-
ent could be involved in specifying an
intermediate zone along the A-V axis
(Weitzel et al., 2003). In ascidians, the
presence of such a gradient has not
yet been investigated.
In Xenopus, maternal VegT mRNA
is localized to the vegetal pole of the
egg. VegT encodes a T-box transcrip-
tion factor and is required for specifi-
cation of the endomesoderm germ
layer in the vegetal hemisphere
(Zhang et al., 1998). However, in
Ciona, there are seven T-box tran-
scription factor genes (one of these is
present as triplicate copies) in the ge-
nome, and none of their maternal
mRNAs are localized to the vegetal
pole (Takatori et al., 2004). This find-
ing is consistent with the observation
that mechanisms involved in specifica-
tion of vegetal fates may differ between
echinoderms/ascidians and amphibi-
ans.
POSTFERTILIZATION
PROCESSES THAT SPECIFY
THE ANTEROPOSTERIOR
AXIS
Cortical Movements
Unfertilized eggs and eggs after the
first phase of ooplasmic segregation
are radially symmetrical along the
A-V axis. This symmetry is broken
during the second phase of ooplasmic
segregation, and eggs become bilater-
ally symmetrical before the first cleav-
age, as cortical and subcortical compo-
nents, such as postplasmic/PEM RNAs
that are located in the vegetal pole
region after the first phase, move to-
ward the future posterior pole (Fig. 3).
This movement is directed by the cen-
trosome introduced by the sperm and
depends on the sperm entry point
(Sardet at al., 1989; Roegiers et al.,
1995). In Phallusia, the sperm tends
to bind to the egg surface at the ani-
mal hemisphere (Speksnijder et al.,
1989) and is conveyed partway toward
the vegetal pole when the plasma
membrane and cortex contracts dur-
ing the first phase, which occurs
shortly after fertilization (Fig. 3). In
contrast to this first phase, which is
driven by microfilaments, the motive
power for the second phase is princi-
pally generated by microtubules of the
sperm aster (Sawada and Schatten,
1988). Vegetally located cortical com-
ponents are then brought to the future
posterior pole together with the sperm
aster, which goes back just beneath
the plasma membrane toward the
equatorial region during the second
phase. The male pronucleus and
sperm aster never cross or go to the
opposite side of the vegetal pole;
therefore, the future posterior side co-
incides with the side of sperm entry
point.
Role of the Posterior-Vegetal
Cytoplasm/Cortex and
Postplasmic/PEM RNAs
The posterior-vegetal cytoplasm/cor-
tex (PVC) after the second phase of
segregation and before the first cleav-
age plays crucial roles in specification
of the A-P axis. Removal of the PVC
(less than 10% of total egg volume)
results in radialization of the cleavage
pattern, producing a mirror image du-
plication of the anterior cleavage pat-
tern in the original posterior half. It
has an effect not only on the cleavage
pattern, but also on the developmen-
tal fates of blastomeres, as they also
show mirror image duplication of the
anterior half. Transplantation of the
PVC to the anterior region brings
about mirror image posteriorization of
the original anterior half (Nishida,
1994). It is worth noting that removal
and transplantation of regional egg
cytoplasm other than the PVC has no
effect on embryogenesis. These micro-
manipulative experiments demon-
strate that some important factors are
localized to the PVC and that no de-
velopmentally important factor is lo-
calized to any small area other than
the PVC. During the second phase,
cER and subcortical mitochondria re-
locate posteriorly (Fig. 3). In contrast,
the plasma membrane and cortical ac-
tin cap do not move. In the past de-
cade, several maternal mRNAs that
are localized to the PVC have been
identified. The first example is the
pem (posterior end mark) mRNA in
Ciona savignyi, which was isolated us-
ing differential screening techniques
with egg fragments prepared by cen-
trifugation (Yoshida et al, 1996). Since
then, nine mRNAs localized to the
PVC have been identified in Halocyn-
thia roretzi (macho-1, Hr-PEM, Hr-
PEM-3, Hr-Wnt-5, Hr-POPK-1, Hr-
GULT, Hr-PEN-1, Hr-PEN-2, Hr-
ZF-1; reviewed in Nishida, 2002a;
Sardet et al., 2005) by the collabora-
tive MAGEST project (cDNA project
of maternal mRNAs: http://www.
genome.jp/magest/; Sasakura et al.,
1998a,b, 2000; Makabe et al., 2001;
Nakamura et al., 2003) and by sub-
traction hybridization screening be-
tween animal and vegetal blastomeres
(Nishida and Sawada, 2001). These
RNAs have been called type I postplas-
mic RNA in Halocynthia (Sasakura et

al., 2000). They are present in cortex of
the PVC. Some of them (macho-1, Hr-
PEM) have been shown to be anchored
to and segregate with the cER (Sardet
et al., 2003). The cER network present
in unfertilized egg provides a scaffold to
anchor these maternal RNAs, and the
second phase of ooplasmic segregation
brings this cER/mRNA domain into an
asymmetric position along the A-P axis.
Localization sequence elements of some
postplasmic RNAs were identified in
their 3⬘-untranslated regions (Sasakura
and Makabe, 2002). Every type I post-
plasmic mRNA shows identical regional
localization during ooplasmic segrega-
tion and cleavage stages (Fig. 3). They
encode variety of protein such as tran-
scription factors, secreted signaling pro-
tein, kinase, RNA-binding protein, and
proteins with no obvious similarity to
known proteins.
What are the functions of type I
postplasmic/PEM RNAs? Removal
and transplantation of the PVC of fer-
tilized eggs, where type I postplasmic
RNAs are localized, and other obser-
vations of events unique to the posterior
region of developing embryos indicate
the following possible functions. The
suppression of functions of some of the
postplasmic RNAs by antisense oligo-
nucleotides supports their roles in pos-
terior specification.
Muscle specification.
A recent molecular study identified
macho-1 mRNA as the localized ma-
ternal determinant of muscle forma-
tion in ascidians (Nishida and
Sawada, 2001; Satou et al., 2002a).
macho-1 mRNA is a type I postplas-
mic RNA, and encodes a Zn-finger pro-
tein that is transcriptional activator
(Sawada et al., 2005). Without ma-
cho-1 function, muscle blastomeres
assume nerve cord fate (Fig. 1; Koba-
yashi et al., 2003).
Responsiveness to inductive
signals.
macho-1 is also involved in generating
difference in responsiveness to induc-
tive signals between notochord and
mesenchyme precursor blastomeres.
macho-1 confers responsiveness to in-
duction of mesenchyme fate by extra-
cellular fibroblast growth factor (FGF)
signal on presumptive mesenchyme
AXIS SPECIFICATION IN ASCIDIANS 1183
blastomeres (Kobayashi et al., 2003),
as discussed later. Thus macho-1 lo-
calization supports central roles in the
specification of cell fates of the poste-
rior blastomeres.
Unequal cleavage.
The centrosome-attracting body (CAB)
was originally discovered as a subcel-
lular structure involved in generation
of unequal cleavages (Hibino et al.,
1998; reviewed in Nishida et al.,
1999). During unequal cleavages in
the posterior-most blastomeres, mi-
crotubule arrays extending from the
posterior centrosome focus on the
CAB, which is present just beneath
plasma membrane at the posterior
pole of the cleaving embryo. Then, as
the microtubule arrays shorten, the
interphase nucleus with the centro-
some shifts posteriorly and ap-
proaches the CAB. Consequently, an
asymmetrically located mitotic appa-
ratus is formed. Unequal division then
takes place, producing a smaller
daughter cell that inherits the CAB is
the posterior-most position in the veg-
etal half of the 16-cell embryo. This
process takes place three times from
the 8- to 64-cell stages, resulting in
three successive unequal cleavages
only at the posterior pole. These un-
equal cleavages make the cleavage
pattern of the posterior-vegetal region
unique, and different from the radial
anterior cleavage pattern. The CAB is
not present in eggs. The PVC of fertil-
ized eggs is required and sufficient for
the formation of the CAB and conse-
quent unequal cleavages (Nishikata et
al., 1999). Results of knockdown of Hr-
PEM mRNA function in Halocynthia
suggest that Hr-PEM is involved in the
positioning of cleavage planes and un-
equal cleavages (T. Negishi, K. Sawada,
and H.N., unpublished observations).
Concentration of postplasmic/
PEM RNAs into the CAB.
Of interest, all postplasmic RNAs are
highly concentrated into a very re-
stricted posterior region of the poste-
rior-vegetal (B4.1) blastomere pair by
the eight-cell stage. This region is in-
deed the CAB. The CAB is rich in ER
(Iseto and Nishida, 1999), and cER
bearing postplasmic RNAs in eggs is
likely to be concentrated into the CAB
during early cleavages (Sardet et al.,
2003). During three rounds of succes-
sive unequal cleavages, the mRNAs
are segregated into the smaller
daughter cells located at the posterior
pole together with the CAB (Fig. 3).
Thus, the CAB serves as the core
structure of a multifunctional complex
that operates cleavage planes and an-
chors postplasmic RNAs. Having both
functions together, the CAB ensures
that postplasmic RNAs are infallibly
partitioned into one of the daughter
cells after cell divisions.
Formation of primordial germ
cells.
An electron microscopic study of 16-
cell embryos revealed that the CAB is
also characterized by an electron-
dense matrix that resembles the germ
plasm in other animals (Iseto and
Nishida, 1999). A pair of the posterior-
most and smallest blastomeres (B7.6
cell pair, brown in Fig. 1) of the 64-cell
embryos that inherit the CAB cease to
divide during early embryogenesis
and give rise to two cells in the
endodermal strand of the tadpole
(Nishida, 1987). In the posterior-most
blastomeres, zygotic gene expression
is repressed (Tomioka et al., 2002), as
observed in Drosophila and C. elegans
germline cells (Seydoux and Dunn,
1997; Van Doren et al., 1998). The ma-
ternal vasa (Ci-DEAD1) mRNA is con-
centrated in the B7.6 cells in Ciona,
and vasa protein also appears in the
descendant cells at tail bud stage (Fu-
jimura and Takamura, 2000; Taka-
mura et al., 2002). After metamorpho-
sis, Vasa-positive cells are found in
the developing gonad. Vasa is a well-
known marker of germ cells in many
animals (Ikenishi, 1998). These obser-
vations support the view that B7.6
cells are primordial germ cells in as-
cidians and that the CAB, enriched in
postplasmic/PEM RNAs, contains
germ plasm.
Specification of trunk ventral
cell precursors.
Precursor blastomeres of trunk ven-
tral cells (B7.5 cells, sister cells of
B7.6 of the 64-cell embryo, purple in
Fig. 1) form in the posterior region of
early embryos (Nishida, 1987). Trunk
ventral cells of tadpole larvae are pro-
1184 NISHIDA
TABLE 1. Key Transcription Factors for Tissue Formation in Ascidian Embryos*
Tissue
Endoderm
Muscle
Notochord
Mesenchyme
Trunk lateral cells
Trunk ventral cells
Brain
Nerve cord
Epidermis
*Expression pattern of each transcription factor is indicated in Fig. 4.
genitors of body wall muscle and heart
of metamorphosed juveniles (Hirano
and Nishida, 1997; Davidson and Le-
vine, 2003). Recently, it was revealed
that a Mesp ortholog in ascidians is
specifically and transiently expressed
in B7.5 cells (Fig. 4) and is essential
for the specification of heart precursor
cells (Satou et al., 2004). Maternal
macho-1 at least is required for Mesp
expression. Other postplasmic/PEM
RNAs might be involved in the speci-
fication of the trunk ventral cells and
zygotic expression of Mesp gene.
Control of cell cycle length.
Cell divisions are retarded in the pos-
terior-vegetal blastomeres compared
with blastomeres in the other regions
(Conklin, 1905). Postplasmic/PEM
RNAs might be also involved in this
retardation of cell cycle.
These events that are unique to the
posterior region and have not been
well analyzed yet, could be attributed
to the postplasmic RNAs that also
have not been analyzed. It is not
known whether the A-P polarity of
eggs is accounted for only by the
asymmetric distribution of the post-
plasmic/PEM RNAs, although the fol-
lowing discussion supports this possi-
bility. Several postplasmic RNAs have
been found as described above, but no
maternal mRNA localized to other re-
gions of eggs has been reported so far.
This finding coincides with the results
of removal and transplantation of egg
cytoplasm other than the PVC.
DNA-binding
Gene domain
Lhx3 Homeobox
TTF1 Homeobox
Tbx6 T-box
Brachyury T-box
Twist-like1 bHLH
Hand-like/NoTrlc bHLH
Mesp bHLH
Otx? Homeobox
?
?
ZYGOTIC GENE
EXPRESSION THAT
ESTABLISHES THE ANIMAL
VS. VEGETAL CELL FATES
Zygotic Genes Downstream
of ␤-catenin
When embryos become multicellular,
cell interactions and zygotic gene ex-
pression start. So far, the earliest zy-
gotic expression in ascidian has been
observed from the eight-cell stage.
Then expression of various genes
starts at the 32-cell stage, correspond-
ing to the mid-blastula stage in ascid-
ians (e.g., Miya and Nishida, 2002).
Several key transcription factors that
show tissue-specific zygotic expression
and are essential for the formation of
each tissue have been identified in as-
cidians in this decade, as described
below (Table 1).
Nuclear ␤-catenin, together with
transcription factor Tcf, promotes zy-
gotic expression of various genes in
the vegetal hemisphere. Genes down-
stream of ␤-catenin have been sur-
veyed in Ciona by subtraction between
embryos overproducing ␤-catenin
and those overproducing cadherin
(Satou et al., 2001a; Imai, 2003) and
by comparison of expression levels by
real-time reverse transcriptase-PCR
for every transcription factor and sig-
naling molecule in the Ciona genome
in early gastrulae injected with a
␤-catenin morpholino antisense oligo-
nucleotide (MO) (Imai et al., 2004).
The downstream genes thus identified
can be classified into three groups.
Reference
Satou et al. [2001a]
Ristoratore et al. [1999]
Mitani et al. [1999]
Yasuo and Satoh [1998]
Imai et al. [2003]
Imai et al. [2003]
Satou et al. [2004]
Wada et al. [2004]
One group represents genes that are
involved in endoderm formation
(FoxA5, Lhx3, TTF1). The second group
includes genes that are relevant to em-
bryonic induction of mesodermal tis-
sues (Fgf9/16/20, chordin, FoxD, Zic,
Brachyury, twist-like1, Mesp). The
genes in the second group are discussed
in the next chapter. Third group con-
tains genes whose functions have not
been fully analyzed in ascidians (cad-
herinII, protochadherin, Eph, lefty, dkk,
DMRT1, hairy, ELK, Fli, jun, msxb,
etc.). Essentially, the expression of
these genes starts during cleavage
stages in the vegetal hemisphere.
Zygotic Genes for
Gastrulation and Endoderm
Fate Specification
The FoxA5 (former forkhead/HNF3␤)
gene is expressed in vegetal blas-
tomeres as early as the 16-cell stage
(Mocu-FH1, Olsen and Jeffery, 1997;
Hr-HNF3, Shimauchi et al., 1997; Ci-
fkh, Di Gregorio et al., 2001). Its expres-
sion is rather broad in the vegetal hemi-
sphere (Fig. 4). When antisense oligo
DNA interfered with the function of Mo-
cuFH1, gastrulation movements were
inhibited (Olsen and Jeffery, 1997). Shi-
mauchi et al. (2001a) suggested a role of
FoxA5 also in notochord formation.
Endoderm precursors are formed
from vegetal pole blastomeres (Fig. 1).
At the 32-cell stage, expression of a
LIM-class homeobox gene, Lhx3, starts
(Wada et al., 1995). Expression of the
gene is not strictly restricted to
endoderm blastomeres at first, but then
Fig. 4. Zygotic gene expression of various developmentally important genes during cleavage stages. Expression of each gene is indicated by blue
dots on blastomeres. The light colors of blastomeres correspond to the colors used in the fate map shown in Figure 1. There are some differences
in the expressions of Tbx6, snail, and Zic genes between Ciona and Halocynthia, although the differences are trivial and not relevant to the discussion
in the review.
1186 NISHIDA
becomes restricted to endoderm blas-
tomeres by the 110-cell stage (Fig. 4).
Satou et al. (2001a) showed that Lhx3 is
required and sufficient for expression of
late marker genes of endoderm differen-
tiation. Overexpression of Lhx3 pro-
motes ectopic endoderm formation
without ␤-catenin activity. Therefore,
Lhx3 would be a key zygotic transcrip-
tion factor for endoderm formation. At
the 64-cell stage, the expression of
TTF1 is initiated exclusively in
endoderm precursors (Fig. 4). TTF1 is a
transcription factor containing an NK-
2–like homeodomain. When synthetic
mRNA of TTF1 is injected into eggs,
ectopic endoderm cells are formed (Ris-
toratore et al., 1999; Satou et al.,
2001a). However, injection of its MO did
not affect endoderm differentiation (Sa-
tou et al., 2001a), although the effi-
ciency of the MO needs to be confirmed.
GATA3/4/5 and Sox17 genes have been
reported to play important roles in
endoderm formation in various metazo-
ans (Laverriere et al., 1994; Hudson et
al., 1997). However, these genes do
not seem to be significant in early
endoderm specification processes in
ascidians to date. Thus, ␤-catenin nu-
clear localization promotes expression
of some zygotic genes that are in-
volved in gastrulation and specifica-
tion of endoderm precursors.
In the animal hemisphere, various
genes also start zygotic expression dur-
ing cleavage stages (e.g., Epi-C in Fig. 4;
Ishida et al., 1996; Miya and Nishida,
2002). However, much attention has not
been paid to the mechanisms of fate
specification of epidermis cells. Thus,
the relationship of these zygotic genes
to ␤-catenin nuclear localization and
their functions in epidermal develop-
ment have not been analyzed. Key tran-
scription factors in epidermal formation
are not yet known.
CELL INTERACTIONS AND
ZYGOTIC GENE
EXPRESSION ALONG THE
ANTEROPOSTERIOR AXIS
Induction of Notochord and
Mesenchyme
Endoderm blastomeres are derived
from the most vegetal region. Meso-
derm and nerve cord precursors sur-
round the central endoderm blas-
tomeres. This marginal area in the
vegetal hemisphere shows high com-
plexity and remarkable asymmetry
along the A-P axis (Fig. 1). The mater-
nal factors in the PVC play crucial
roles in making this region asymmet-
ric. In addition, inductive cell interac-
tions contribute to generating cell di-
versity in this area. The importance of
cell interactions has been revealed by
blastomere isolation and recombina-
tion and with inhibitors of cell signal-
ing (reviewed by Nishida, 2002a,b and
references therein). Thus, it is now re-
alized that the development of ascid-
ian embryos is not entirely mosaic as
has been hitherto thought. Key terms
to understand what happens in this
area are “FGF signaling,” “directed
signaling and asymmetric division,”
and “cell responsiveness.”
FGF Signaling
In the 32-cell embryos, central
endoderm blastomeres are flanked by
a single circle of marginal blastomeres
(Fig. 1). The endoderm blastomeres
emit an inductive signal toward these
marginal cells. FGF (an Fgf9/16/20
gene product) plays a major role as the
signaling molecule (Nakatani et al.,
1996; Kim et al., 2000; Satou et al.,
2002c; Imai et al., 2002a). Treatment
of isolated blastomeres with recombi-
nant FGF protein efficiently mimics
the normal cell responses. Fgf9/16/20
MO suppresses the inductions. Ex-
pression of the Fgf9/16/20 gene starts
at the 16-cell stage (Fig. 4) and occurs
downstream of ␤-catenin (Imai et al.,
2002a). Thus, acquisition of the induc-
ing ability depends on the position of
blastomeres along the A-V axis. The
inductive interactions take place at
the 32-cell stage. Results of experi-
ments involving recombination of iso-
lated blastomeres at various stages
and of experiments determining the
periods of sensitivity to FGF treat-
ment and to signaling inhibitors all
support the idea that major processes
of induction take place at the 32-cell
stage (Nakatani et al., 1996; Kim and
Nishida, 2001). The expression of
Fgf9/16/20 occurs at the right time
and in the right place. The signal-re-
ceiving blastomeres quickly lose their
competence at the next cleavage (Na-
katani and Nishida, 1999).
Directed Signaling and
Asymmetric Division
Then asymmetric cell divisions occur
in the marginal blastomeres. These
blastomeres divide radially so that
two lines of blastomeres encircle the
endoderm blastomeres at the 64-cell
stage (Fig. 1). Four anterior blas-
tomeres of the 32-cell embryo divide
into four outer nerve cord precursors
(colored pale blue in Fig. 1) and four
inner notochord precursors (pink) of
the 64-cell embryo. (The central ner-
vous system of ascidian tadpole larvae
consists of an anterior brain in the
cranial region and a posterior nerve
cord in the trunk and tail region [Fig.
1]. Posterior-nerve cord precursors are
blastomeres in the vegetal hemi-
sphere and give rise to the posterior
neural tube in the trunk and tail re-
gion.) Similarly in the posterior re-
gion, four blastomeres of the 32-cell
embryo divide into four outer muscle
precursors (red) and four inner mes-
enchyme precursors (dark green). In
these processes, outer nerve cord and
muscle fates represent the default
state and inner notochord and mesen-
chyme fates represent the induced
fates. When the relevant blastomeres
are isolated before induction at the
32-cell stage or treated with signaling
inhibitors, both daughters of them as-
sume the default fate. In contrast,
when isolated blastomeres are treated
with FGF dissolved in seawater, both
daughters take the induced fate (Kim
et al., 2000; Minokawa et al., 2001).
A conspicuous feature of these in-
ductions is the asymmetric cell divi-
sions of the cells that received the in-
ductive signal. On the basis of the
results described above, it is possible
to imagine the following scenario. In
normal embryos, the precursor cells
of the 32-cell embryo receive an
endoderm signal from the vegetal
pole, and only one of the daughter
cells facing the endoderm assumes an
induced cell fate in the 64-cell embryo.
Directed signals that emanate from
endoderm blastomeres may polarize
the responding blastomeres at the 32-
cell stage and promote asymmetric di-
visions that operate in both the ante-
rior and posterior regions. This
directed signaling and asymmetric di-
vision model is supported by the fact
that treatment of isolated blastomeres

with FGF in seawater causes both
daughters to assume induced fates,
because isolated mother blastomeres
receive the signal over the entire cell
surface (Kim et al., 2000; Minokawa et
al., 2001). The results of blastomere
transplantation also support the
model (G.J. Kim and H.N., unpub-
lished). Presumably, FGF signaling
causes localized changes in the
mother cell. The extracellular FGF
signal is transduced to the nuclei by
conserved molecules in ascidians:
FGF receptor, ras, MEK, and MAPK
(Nakatani and Nishida, 1997; Kim
and Nishida, 2001; Shimauchi et al.,
2001b). The mRNAs of these genes are
supplied maternally. The simplest
model is that MAPK is activated in
one side of the signal-receiving cell
that faces the inducer cell, and then
cleavage starts before the activation
propagates into the entire cytoplasmic
region of the cell. At least, activated
and phosphorylated MAPK is ob-
served only in the nucleus of one
daughter cell that faces the endoderm
(Nishida, 2003), although a high back-
ground of immunostaining unfortu-
nately prevented detection of asym-
metric activation of MAPK in the
cytoplasm of the mother cells before
the asymmetric cell division.
Responsiveness to Inductive
Signal
Ets family transcription factors is
known as one of the targets of the
Ras–MAPK signaling pathway (re-
viewed by Wasylyk et al., 1998; Yordy
and Muise-Helmericks, 2000). It has
been shown that Ets is also required
for notochord and mesenchyme induc-
tion in ascidian (Miya and Nishida,
2003). Ets mRNA is maternally
present and ubiquitous. The same
FGF signal and the same intracellular
signal transduction cascade down to
the Ets transcription factor induce dif-
ferent responses in the anterior and
posterior marginal blastomeres. What
causes this difference in responsive-
ness? The difference must preexist
within the responding cells. Results of
removal and transfer of egg cytoplasm
indicate that the PVC is involved. In-
deed, macho-1 causes the difference
between anterior and posterior blas-
tomeres (Kobayashi et al., 2003). ma-
cho-1– deficient embryos form noto-
AXIS SPECIFICATION IN ASCIDIANS 1187
chord in the posterior region in place
of mesenchyme. macho-1– overex-
pressed embryos form mesenchyme in
place of notochord. Most intriguing re-
sult is that when macho-1 is overex-
pressed in entire embryos and blas-
tomeres of the animal hemisphere are
isolated and treated with FGF, those
cells develop into mesenchyme. ma-
cho-1 is sufficient to confer compe-
tence for induction by FGF to form
mesenchyme even in animal blas-
tomeres. Therefore, without macho-1,
the anterior marginal blastomeres di-
vide asymmetrically into notochord
and nerve cord precursors. The poste-
rior marginal blastomeres containing
macho-1 divide into mesenchyme and
muscle.
In inductive interactions, extracel-
lular signaling molecule and intrinsic
factors are combined in signal-receiv-
ing cells, conferring particular cell
fates. Intrinsic factors of responding
cells play roles in defining the re-
sponse. Thus, in the case of these as-
cidian embryonic inductions, the ex-
tracellular signal is transduced into
the Ets transcription factor, while the
intrinsic competence factor is macho-1
transcription factor.
Zygotic Expression of Key
Transcription Factors:
Muscle and Notochord
macho-1 is a transcription activator
and binds to specific DNA sequences.
Yagi et al. (2004) and Sawada et al.
(2005) have reported genes down-
stream of macho-1. Various genes for
transcription factors and muscle
structural protein have been found.
Some of these genes have a macho-
binding consensus sequence in their 5⬘
upstream regions (Yagi et al., 2004).
Among these genes, Tbx6 is a candi-
date of a key transcription factor for
muscle development, because misex-
pression of Tbx6 causes ectopic ex-
pression of muscle structural genes
(Mitani et al., 1999). Tbx6 is expressed
in muscle precursor blastomeres at
cleavage stages (Fig. 4). The expres-
sion of a myogenic factor (AMD1 in
Halocynthia, Ci-MDF in Ciona; Satoh
et al., 1996; Meedel et al., 1997) is also
muscle-specific at the cleavage stages,
although the function of it is not fully
analyzed yet. On the other hand,
twist-like1 is a key transcription fac-
tor for mesenchyme differentiation; it
is specifically expressed in mesen-
chyme precursors. The expression de-
pends on macho-1 and FGF, and
twist-like1 is essential for the expres-
sion of late mesenchyme marker
genes (Fig. 4; Imai et al., 2003; To-
kuoka et al., 2004). snail also lies
downstream of macho-1 (Kobayashi et
al., 2003) and is involved in the control
of responsiveness. snail encodes a Zn-
finger transcription repressor and is
expressed in muscle and mesenchyme
precursors at the 32-cell stage (Fig. 4;
Erives et al., 1998; Wada and Saiga,
1999). As discussed two paragraphs
below, brachyury is a key transcrip-
tion factor for notochord differentia-
tion in ascidians. Analysis of the cis-
regulatory region of the brachyury
gene and overexpression of snail
mRNA indicate that snail represses
the activation of brachyury in the pos-
terior blastomeres (Fujiwara et al.,
1998; Kobayashi et al., 2003). This
finding partly explains how the ma-
cho-1 transcription activator can sup-
press notochord fate in the posterior
region.
Antibodies against macho-1 protein
are not available yet, but evidence is
accumulating that macho-1 transla-
tion is initiated at the eight-cell stage,
and the protein diffuses into the entire
cytoplasm of the posterior-vegetal
(B4.1) blastomeres of the eight-cell
embryo. The B4.1 blastomere gives
rise mainly to muscle, mesenchyme,
and endoderm. However, every de-
scendant of the B4.1 blastomere, even
endoderm-lineage cells, assumes mus-
cle fate without cell interaction (Kon-
doh et al., 2003). FGF and bone
morphogenetic protein (BMP) are in-
volved in formation of endoderm cells
from the B4.1 blastomeres. By con-
trast, endoderm formation from the
A4.1 (anterior-vegetal) blastomeres,
which do not have macho-1 products,
is a cell-autonomous process and does
not require cell interaction. The cell
interactions in the B4.1 descendants
are necessary for the suppression of
macho-1 function in B-line endoderm
precursors, because injection of ma-
cho-1 MO makes cell interaction un-
necessary (Kondoh et al., 2003).
Therefore, macho-1 directs muscle
fate in posterior muscle cells; its func-
tion is likely modified by FGF signal-
ing to promote mesenchyme fate in
1188 NISHIDA
intermediate mesenchyme cells; and
in central B-line endoderm cells its
function is totally suppressed by FGF
and BMP. Probably there are intrinsic
differences in responsiveness to FGF
between mesenchyme and endoderm
blastomeres, and the localization of
endoderm determinants could account
for the differences. The cell interac-
tions would restrict the regions where
maternal determinants work, and
play a role in marking a precise
boundary between precursor cells of
different tissue types. It would be
hard for embryos to precisely partition
muscle determinants exclusively into
multiple muscle precursor blas-
tomeres through a complicated cleav-
age history. These findings suggest
that the cell interactions are also used
to suppress functions of inappropri-
ately distributed maternal determi-
nants after embryogenesis starts.
FGF treatment can induce noto-
chord fate only in presumptive noto-
chord/nerve cord-lineage cells. Cells in
the animal hemisphere never respond
to FGF by assuming notochord fate.
This finding indicates that responsive-
ness to induction to form notochord is
restricted to marginal notochord/
nerve cord-lineage cells. ␤-catenin–
deficient embryos fail to develop noto-
chord, as the downstream FGF9/
16/20 is not expressed. However, even
if FGF9/16/20 is overexpressed in
␤-catenin– deficient embryos, noto-
chord is not formed (Imai et al., 2000,
2002a). This indicates that compe-
tence to be induced to form notochord
is also lost. It has been suggested that
␤-catenin, FoxD, and Zic are involved
in the acquisition of competence (Fig.
4). MOs of these genes abolish noto-
chord. FoxD gene is a direct target of
␤-catenin, as its expression depends
on ␤-catenin and on a Tcf-binding site
in its 5⬘ upstream cis-element (Imai et
al., 2002b). FoxD expression is inde-
pendent of FGF9/16/20 and vice
versa. Expression of FoxD is transient
at the 16-and 32-cells stages in the
vegetal hemisphere. FoxD is expressed
in notochord/nerve cord/endoderm-
lineage blastomeres at the 16-cell
stage. Then Zic expression starts in
notochord/nerve cord-lineage cells at
the 32-cell stage (Wada and Saiga,
2002; Imai et al., 2002c). The expres-
sion depends on FoxD and persists in
both notochord and nerve cord precur-
sors at the 64-cell stage. Finally, ex-
pression of brachyury, a key transcrip-
tion factor in notochord formation,
specifically starts in notochord precur-
sors of 64-cell embryos (Yasuo and
Satoh, 1998). Injection of Zic MO
eliminates brachyury expression and
notochord formation (Wada and Saiga,
2002; Imai et al., 2002c), and Zic over-
expression ectopically activates the
brachyury promoter through the Zic-
binding site (Yagi et al., 2003). Zic ap-
pears to be a competence factor in no-
tochord induction, although this needs
further investigation. Zic contains a
Zn-finger domain that is closely re-
lated to that of macho-1. Therefore, it
is interesting that similar Zn-finger
proteins with the same origin are used
in anterior (Zic) and posterior (ma-
cho-1) regions. Another interesting
symmetry is seen in the key transcrip-
tion factors. Both brachyury and Tbx6
belong to the T-box family, and they
participate in notochord (anterior)
and muscle (posterior) formation, re-
spectively.
Zygotic Expression of Key
Transcription Factors:
Mesenchyme, Trunk Ventral
Cells, and Trunk Lateral
Cells
Among mesodermal tissues, muscle
and notochord function at the swim-
ming tadpole stage. In addition, three
cell types in the larvae—mesenchyme,
trunk lateral cells (TLCs), and trunk
ventral cells (TVCs)—are preserved in
the larvae and function after meta-
morphosis. Mesenchyme gives rise to
tunic cells in the juvenile. TLCs are
precursors of blood cells and body wall
muscle. TVCs develop into heart and
body wall muscle (Hirano and
Nishida, 1997). As mentioned above,
mesenchyme specification requires
macho-1 and FGF. Twist-like1 is a key
transcription factor for mesenchyme.
Recent studies have shown that TLCs
and TVCs are specified during cleav-
age stages. TLC precursor (A7.6) blas-
tomeres of the 64-cell embryo form in
the lateral region of the vegetal hemi-
sphere (sky blue in Fig. 1). Hand-like/
NoTrlc is a key transcription factor for
TLC formation (Imai et al., 2003). It
has a basic helix–loop– helix (bHLH)
domain and is specifically expressed
in TLC precursors from the 64-cell
stage (Fig. 4). FoxD activity is re-
quired for the expression of the Hand-
like gene. Blastomere isolation and re-
combination experiments showed that
inductive interaction from the animal
blastomere at the 16-cell stage is impor-
tant and that only TLC-lineage blas-
tomeres have competence (Kawami-
nami and Nishida, 1997).
TVC precursor blastomeres (B7.5)
and putative germline precursor blas-
tomeres (B7.6) are present in the pos-
terior-most region (purple and brown
in Fig. 1, respectively). B7.5 and B7.6
are sister cells that are generated by
the final unequal cleavage to the 64-
cell stage. B7.6 is the smaller blas-
tomere; it is present at the posterior
pole and inherits the CAB and post-
plasmic/PEM RNAs as mentioned be-
fore. On the other hand, B7.5 is pre-
cursor of TVCs of the larvae and adult
heart. Mesp, which has a bHLH do-
main, is a key transcription factor for
formation of TVCs and adult heart. It
is specifically expressed in TVC pre-
cursors from the 110-cell stage (Fig.
4). macho-1 is required for Mesp ex-
pression (Satou et al., 2004). In accor-
dance, every descendant of B7.5 as-
sumes larval muscle fate without cell
interaction (Nishida, 1992a; Kondoh
et al., 2003). In more detail, the B7.5
blastomere divides into a larval mus-
cle precursor and a TVC precursor at
the next division. Therefore, directed
signaling and asymmetric division
might also operate in this cell division
(Fig. 1). Various cell interactions are
speculated to be involved in TVC spec-
ification (Davidson and Levine, 2003).
The competence factor for TVC induc-
tion may be one of the products of
postplasmic/PEM RNAs.
The marginal zone of the vegetal
hemisphere is patterned along the A-P
axis, as discussed above. Localized
maternal factors are involved in the
A-P polarization. Then, cellular inter-
actions play important roles in this
patterning when embryos become
multicellular. Zygotic gene expression
is involved in production of both of
inductive signals and competence fac-
tors, and finally expression of cell-type
specific key transcription factors is
bought about.
 AXIS SPECIFICATION IN ASCIDIANS 1189

Animal Hemisphere have been analyzed blastomere by communication at early developmental

blastomere at the single-cell level in stages because of its limited cell num-
What happens in the animal hemi-
the ascidian embryo. This analysis ber and invariant mode of development.
sphere is much simpler. Most regions
combined with the stereotypic cleav-
of the animal hemisphere are occupied
age pattern makes studies of ascidian
by epidermis blastomeres. Only on the
embryogenesis easy to analyze and
Maternal Factors
anterior edge are brain precursors
understand. Ascidian embryogenesis Analysis of maternal processes is also
formed (Fig. 1). The formation of brain
has been hitherto regarded as a typi- under way, and the most pressing
precursors requires induction from
cal example of mosaic development, questions are: How is the A-V polarity
the vegetal blastomeres (reviewed by
but recent analyses revealed that cell established during oogenesis? Is Di-
Okamura et al., 1993; Lemaire et al.,
interactions play equally important shevelled a vegetal determinant in as-
2002; Meinertzhagen et al., 2004).
roles. In retrospect, there are two cidians? Is there a gradient of ␤-cate-
Otx, a putative key transcription fac-
main reasons for ascidian embryos to nin stabilization along the A-V axis,
tor for brain development, is ex-
be regarded as a mosaic. When blas- and how is the intermediate marginal
pressed in these blastomeres (Fig. 4).
tomeres are isolated at an early cleav- zone specified? What are the roles of
Otx is a homeobox transcription factor
age stage, for example at the eight-cell unanalyzed postplasmic/PEM RNAs
that plays important roles in brain de-
stage, cell interactions take place in establishment of the A-P axis? Cel-
velopment in Drosophila and in verte-
within the partial embryos derived lular mechanisms of unequal cleavage
brates (Simeone, 1998). The functions
from the isolated blastomeres. There- in relation to the CAB and anchoring
of otx have been investigated with MO
fore, the blastomeres can develop var- of postplasmic RNA to cER will be im-
(Wada et al., 2004). In the absence of
ious tissues as expected from the fate portant issues to pursue. In addition,
Otx function, brain development was
map, and spuriously behave in an au- spatiotemporal analysis of FGF signal
abrogated but expression of some
tonomous way. In other words, the transduction processes will be re-
brain-specific genes persisted. There-
blastomeres can develop autono- quired to fully understand the mecha-
fore, the idea that Otx is a key tran-
mously in isolation; however, tissue nisms of asymmetric divisions after
scription factor is still controversial.
formation is not cell-autonomous pro- cells receive directed signals.
FGF/Ets and GATA are crucial for the
cess in these cases.
expression in brain precursors (In-
The second reason is that ascidian
azawa et al., 1998; Miya and Nishida, Genome and ESTs
embryos do not compensate for lost
2003; Bertrand et al., 2003). In accor-
parts if blastomeres are ablated. This An evolutionary point of view is also
dance, Ets- and GATA-binding cis-el-
is because competence or responsive- interesting because of the phylogenic
ements are crucial for the expression
ness to be induced to certain tissues is position of ascidians (Di Gregorio and
of the Otx–reporter construct. Thus,
indeed restricted to the tissue precur- Levine, 2001; Corbo et al., 2001; Sa-
the FGF signal from the vegetal hemi-
sor blastomeres, as discussed in this toh, 2003a). So far, most research has
sphere induces brain precursors
article. Generally in animal develop- been concentrated on embryogenesis.
through activation of Ets. GATA gene
ment, various organisms use a limited However, analysis of mechanisms of
expression is maternal and ubiqui-
repertoire of signaling molecules for metamorphosis is also worth analysis
tous, but it is translated only in ani-
intercellular communication. The same (Hirano and Nishida, 1997, 2000; Eri
mal blastomeres at the late 32-cell
signals are used over and over again et al., 1999; Davidson and Swalla,
stage, when induction takes place.
in different cells at different stages, 2002).
These results suggest that GATA is
with different biological outcomes de- Recently, infrastructure for ascid-
the competence factor that restricts
pending on the spatial and temporal ian research has been set up expedi-
brain induction to the animal hemi-
context. To understand animal devel- tiously. A cDNA project involving
sphere (Bertrand et al., 2003). It is
opment, it is important to clarify how large-scale ESTs and in situ hybrid-
still not clear how the brain is induced
embryonic cells can respond differ- ization of maternal mRNA in Halocyn-
only at the anterior edge, although
ently to the same signal, and to under- thia roretzi (http://www.genome.jp/
blastomere recombination experi-
stand how competence is restricted to magest/; Makabe et al., 2001) and a
ments and treatment with inducing
specific cells. cDNA project on maternal and zygotic
substance demonstrated that the an-
Ascidian embryo provides a good transcripts in Ciona intestinalis (http://
terior and posterior blastomeres have
model for elucidating this issue. Cel- ghost.zool.kyoto-u.ac.jp/indexr1.html;
different efficiencies of brain induc-
lular simplicity and transparency of Satou et al., 2001b, 2002b) will help fu-
tion (Okado and Takahashi, 1993;
ascidian embryos have opened the ture studies. The genome sequence of C.
Hudson and Lemaire, 2001).
way to the use of sophisticated labeling intestinalis is completed (http://genome.
and imaging techniques, which allow jgi-psf.org/ciona4/ciona4.home.html; De-
SUMMARY AND FUTURE for three-dimensional and four-dimen- hal et al., 2002; Satoh, 2003b), and that
DIRECTIONS sional reconstruction of virtual em- of C. savignyi is nearly completed (http://
Mosaic Development and bryos. Hence, the imaging tools may www.broad.mit.edu/annotation/ciona/;
help access to cell contact surface or cell http://www2.bioinformatics.tll.org.sg:
Cell Interactions division features of neighboring blas- 8082/Ciona_savignyi/). Comparison of
As can be seen in Figures 1, 3, and 4, tomeres. Ascidian embryo is indeed a promoter sequences in these closely
fate specification and gene expression perfect system for elucidating cellular related species are useful for detecting
1190 NISHIDA
conserved regulatory cis-elements for
gene expression (Johnson et al., 2004).
Developmentally important genes
are annotated in a special ascidian is-
sue of Development Genes and Evolu-
tion (Volume 213, No. 5– 6, 2003). Re-
cently, Imai et al. (2004) listed every
gene for transcription factors and sig-
naling molecules in the C. intestinalis
genome, and the expression pattern
during embryogenesis was compre-
hensively reported (http://ghost.zool.
kyoto-u.ac.jp/tfst.html). The minimal
gene redundancy in the ascidian ge-
nome will simplify our understanding
of the expression patterns and the re-
sults of gene knockdown experiments.
The wealth of genetic information on
ascidians will promote studies to en-
visage the molecular network during
ascidian embryogenesis. Genetic ap-
proaches are now feasible in Ciona
and have begun bearing fruit (e.g.,
Nakatani et al., 1999; Awazu et al.,
2004). Another possible genetic ap-
proach will be to use appendicularians
(Urochordata, a larvecian, Oiko-
pleura), which have a simple chordate
body plan (Fenaux, 1998; Nishino and
Satoh, 2001), and their life cycle is
only 4 to 7 days.
ACKNOWLEDGMENTS
I thank Dr. G. Kumano, Dr. J. Matsu-
moto, and Dr. C. Sardet for their valu-
able comments on the manuscript.
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