Review articles
Patterning the marginal zone of
early ascidian embryos: localized
maternal mRNA and inductive
interactions
Hiroki Nishida
Summary
Early animal embryos are patterned by localized egg
cytoplasmic factors and cell interactions. In invertebrate
chordate ascidians, larval tail muscle originates from the
posterior marginal zone of the early embryo. It has
recently been demonstrated that maternal macho-1
mRNA encoding transcription factor acts as a localized
muscle determinant. Other mesodermal tissues such as
notochord and mesenchyme are also derived from the
vegetal marginal zone. In contrast, formation of these
tissues requires induction from endoderm precursors at
the 32-cell stage. FGF–Ras–MAPK signaling is involved
in the induction of both tissues. The responsiveness for
induction to notochord or mesenchyme depends on the
inheritance of localized egg cytoplasmic factors. Pre-
vious studies also point to critical roles of directed
signaling in polarization of induced cells and in subse-
quent asymmetric divisions resulting in the formation
of two daughter cells with distinct fates. One cell ado-
pts an induced fate, while the other assumes a default
fate. A simple model of mesoderm patterning in asci-
dian embryos is proposed in comparison with that of
vertebrates. BioEssays 24:613–624, 2002.
ß 2002 Wiley Periodicals, Inc.
Introduction
Recent research using ascidian embryos has yielded insight
into the mechanisms that mediate fate specification during
early embryogenesis. Ascidians are simple chordates (Uro-
chordata, Ascidiacea) and their embryogenesis shows the
Department of Biological Sciences, Tokyo Institute of Technology,
Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
E-mail: hnishida@bio.titech.ac.jp
Funding agencies: JSPS, HFSP, The Ministry of Education, Sports
and Culture of Japan.
DOI 10.1002/bies.10099
Published online in Wiley InterScience (www.interscience.wiley.com).
Abbreviations: macho-1, maboya no cho omoshiroi idenshi-1 in
Japanese; EST, expression sequence tag; MAPK, mitogen-activated
protein kinase; ERK, extracellular signal-regulated kinase; MEK,
mitogen-activated protein kinase/ extracellular signal-regulated
kinase kinase: FGF, fibroblast growth factor; PVC, posterior-vegetal
cytoplasm
BioEssays 24:613–624, ß 2002 Wiley Periodicals, Inc.
following characteristics (Fig. 1).(1–6) (1) Eggs develop into
tadpole larvae that have the basic body plan of chordates,
characterized by a dorsal neural tube and tail containing a
notochord flanked by bilateral muscle tissue.(7) (2) Their
organization is much simpler than that of a vertebrate. They
consist of a small number of cells and cell types, and cell
lineages are invariant among individuals. (3) Fate restriction of
blastomeres to a single cell type is almost completed as early
as the 110-cell stage.(8) (4) Precursor blastomeres of several
cell types show cell-autonomous development which led to the
term ‘mosaic’ development more than a century ago.(9) Thus,
ascidian embryogenesis is characterized by simplicity, a
feature that may enable the mechanisms of cell fate specifi-
cation for the entire embryo and for every cell type at both the
cellular and molecular level to be understood.
Recent molecular analysis together with significant accu-
mulation of knowledge revealed by classic experimental
embryology have advanced our understanding of how devel-
opmental fates of blastomeres are specified in early ascidian
embryos. Large-scale EST analysis accompanied by descrip-
tions of the expression patterns of each gene has been carried
out,(10,11) and, currently, genome sequencing projects are
underway. Thus the ascidian is being recognized as a model
organism in developmental biology. This article reviews the
mechanisms of fate specification of mesodermal tissues in the
marginal zone, emphasizing the involvement of localized
maternal mRNA and inductive cell interactions.
Mesodermal tissues originate from the
marginal zone of the vegetal hemisphere
As in frog embryos, ectoderm, mesoderm and endoderm
territories are present in this order along the animal–vegetal
axis of the fate map (see Fig. 4). Most animal blastomeres give
rise to epidermis whose fate is specified by an unknown
localized ooplasmic factor.(12) The anteriormost region of the
animal hemisphere develops into brain through formation of
the neural tube, and inductive interactions are required for
brain formation.(13) Endoderm is derived from the vegetal pole
region, whose fate is also specified by an unknown localized
ooplasmic factor.(14) A recent study has indicated that b-
catenin plays an important role in endoderm fate specification
in ascidians.(15) b-catenin signaling pathway plays crucial
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Figure 1. Embryogenesis of the ascidian, Halocynthia roretzi. A: Adult animal. The size is approximately 15 cm. (courtesy of Dr.
T. Numakunai) B: Fertilized egg. The diameter is 280 mm. C: 64-cell-stage embryo just before start of gastrulation. Vegetal view;
anterior is up. D: Neurula. Anterior is to the right. Neural tube is closing from the posterior side. E: Initial tailbud. Each constituent cell is
still visible. F: Tadpole larva just before hatching, 35 h after fertilization. It consists of approximately 3000 cells.

roles in maternal mechanisms that specify the dorsal–ventral
axis in amphibian and fish,(16–18) and animal–vegetal axis
in sea urchin.(19–21) In this regard, ascidian embryos show
similarity to echinoderm embryos.
The marginal zone of the vegetal hemisphere, which
surrounds the central endodermal area, is mesodermal
territory. The major mesodermal tissues are muscle, noto-
chord and mesenchyme. Mesenchyme cells are preserved in
the larvae, and give rise to tunic cells after metamorphosis.(22)
The minor mesodermal tissues consist of trunk lateral cells
and trunk ventral cells. They are precursor cells of the body
wall muscle, heart and blood cells of the metamorphosed
juvenile.(22)
Localization of muscle determinants in the
egg cytoplasm
Maternal information stored in particular regions of the egg
cytoplasm plays an important role in the determination of
developmental fates during early animal development. The
partitioning of colored egg cytoplasm into specific lineage
blastomeres,(9) the autonomous differentiation of isolated and
dissociated blastomeres,(23,24) and the results of transplanta-
tion of ooplasm from specific regions(3) have revealed the
presence and localization of maternal determinants in the
ascidian. These localized maternal determinants, which
specify epidermis, muscle and endoderm fates, have provided
an understanding of the mosaic manner of development.
A great deal of interest has been concentrated on
mechanisms underlying the formation of muscle cells in the
larval tail, since Conklin reported in 1905 that yellow-colored
myoplasm in the eggs of some species is preferentially
segregated into muscle-lineage blastomeres.(9) Ooplasmic
transplantation experiments have indicated that muscle deter-
minants are present as a gradient in unfertilized eggs with
the highest activity at the vegetal pole (Fig. 2A). Just after
fertilization, these determinants are concentrated at the
vegetal pole, and then move to the future posterior pole during
ooplasmic segregation. Thus, they settle at sites that corres-
pond to the appropriate region in the future fate map before
cleavage starts. Muscle determinants are partitioned into
muscle progenitor blastomeres during subsequent clea-
vages.(25,26) The distribution of cytoplasm that promotes
muscle formation coincides with that of Conklin’s myoplasm,
which is a cytoplasmic domain enriched in mitochondria, endo-
plasmic reticulum , cytoskeleton, and pigment granules.(27,28)
macho-1 maternal mRNA as a
muscle determinant
A recent molecular study has identified a strong candidate for
the localized maternal determinants of muscle formation in the

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Figure 2. Distribution of cytoplasmic determinants and macho-1 maternal mRNA in eggs. A: Distribution of muscle determinant during
ooplasmic segregation revealed by cytoplasmic transplantation experiments. Animal pole is up and vegetal pole is down. Anterior is to
the left and posterior is to the right. B: Distribution of maternal macho-1 mRNA shown by in situ hybridization at stages corresponding to
A. Ani, animal pole. Veg, vegetal pole. A, anterior. P, posterior. (From Nishida H, and Sawada K. Nature 2001;409:724–729, with
permission of Macmillan Magazines Ltd.)(29)

ascidian, Halocynthia roretzi.(29) In that study, a macho-1
cDNA clone was isolated by subtraction hybridization screen-
ing between the animal and vegetal hemispheres of the 8-cell
embryo, and the distribution of maternal macho-1 mRNA in
eggs (Fig. 2B) was found to correspond closely to the
distribution of muscle determinants (Fig. 2A). Maternal
macho-1 mRNA was then depleted by injection of antisense
phosphorothioate oligodeoxynucleotides. The macho-1-de-
pleted eggs showed normal ooplasmic segregation and
cleavages. The eggs underwent gastrulation, and embryo-
genesis appeared normal up to the neurula stage. However, in
tailbud embryos, tail formation was severely affected. At
hatching, the trunk region appeared normal but the tail was
shortened (Fig. 3A,B). The formation of most tissues in
macho-1-depleted larvae, epidermis, sensory pigment cells,
notochord and endoderm, was normal. However, the tail
muscle cells were greatly reduced as shown by monitoring the
expression of the muscle markers myosin, acetylcholinester-
ase, and actin (Fig. 3C,D,H,I). Although muscle was reduced,
some muscle cells were always present at the tip of the tail.
There are two types of muscle cell in the larval tail: primary and
secondary muscle cells. Formation of the primary muscle
shows cell autonomy, and its fate is specified by localized
muscle determinants. In contrast, the secondary muscle cells
located at the tip of the tail are specified through cell inter-
actions during gastrulation.(30,31) Experiments involving isola-
tion of the primary muscle precursor blastomeres in macho-1-
depleted embryos indicated that only primary muscle cells
were lost (Fig. 3E,F). Injection of synthetic macho-1 mRNA
into macho-1 deficient embryos restored muscle formation.
In a further experiment, injection of synthetic macho-1
mRNA caused ectopic muscle formation in non-muscle-
lineage cells (Fig. 3G,J). These results indicate that macho-1
is both required and sufficient for specification of muscle fate.
However, these criteria are not enough to confirm conclusively
that macho-1 is the localized muscle determinant. For
example, in the frog egg, b-catenin is required and sufficient
for promoting the development of dorsal structures. But when
the dorsal cytoplasm of b-catenin-depleted eggs is transferred
to the ventral side of the intact egg, a secondary dorsal axis is
still induced.(32) This observation indicates that b-catenin
functions as a component of the machinery transducing the
dorsal determinant, but is not the dorsal determinant itself.
A similar experiment was carried out to test macho-1. The

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Figure 3. Depletion and overexpression of maternal macho-1 mRNA. A: Larvae injected with control oligo. In lower photo, tail muscle
cells are stained with myosin antibody. B–D: Larvae injected with antisense oligo of macho-1 mRNA. B: Morphology. Tail is shrunken.
C: Tail muscle cells that express myosin are reduced. D: Tail muscle cells detected by acetylcholinesterase histochemistry. E: Precursor
blastomere of primary muscle cells (B4.1 cells) was isolated from control embryo and cultured as a partial embryo. Myosin is expressed.
F: Injection of antisense oligo results in complete loss of myosin expression. G: After injection of synthetic macho-1 mRNA into eggs,
the embryos develop into aberrant larvae and excess muscle forms within whole embryos. H: Muscle actin gene expression in primary
muscle lineage cells of the 110-cell embryo. I: Expression of muscle actin is reduced in macho-1-deficient embryos. J: Injection of
synthetic mRNA causes ectopic expression of actin genes. K: After injection of FLAG-tagged macho-1 mRNA, the protein is present in
nuclei at the 110-cell stage. From Nishida H, and Sawada K. Nature 2001;409:724–729, with permission from Macmillan Magazines
Ltd.(29)

posterior-vegetal cytoplasm of the fertilized egg has the ability
to promote muscle formation when transferred into epidermis
blastomeres. By contrast, the cytoplasm of macho-1-depleted
eggs did not promote ectopic muscle formation in epidermis
blastomeres. Thus maternal macho-1 mRNA satisfies the key
criteria for the localized muscle-forming factor in ascidian
eggs, whose existence was first proposed by Conklin one
century ago.
The macho-1 gene shows no zygotic expression. The
macho-1 protein has five CCHH-type zinc-finger repeats in the
central part that have similarity with Zic, GLI, and odd-paired
proteins. All of these proteins are transcription factors.(33–35)
As macho-1 protein synthesized from FLAG-tagged mRNAs
accumulates in the nuclei during the cleavage stage (Fig. 3K),
macho-1 is most likely a transcription factor.
macho-1 may directly control the expression of muscle
structural genes such as the actin and myosin genes during
the initial process of muscle formation because these are
activated as early as the 32-cell stage.(36) macho-1 may also
promote the expression of regulatory genes such as ascidian
Tbx6(37,38) and myogenic factor.(39,40) In ascidians, the expres-
sion of these regulatory factors occurs a little later, after the
initiation of expression of the muscle structural genes. There-
fore, it is suggested that these regulatory factors cooperate
together to maintain muscle differentiation processes utilizing
T-box-protein-binding sites and E-boxes in controlling ele-
ments of the muscle structural genes,(41) after the initial
process has been triggered by macho-1.
Cell interactions in ascidian embryos
Cell interactions, especially inductive interactions, play crucial
roles in animal embryogenesis.(42,43) During the last decade,
there have been tremendous advances in understanding
the various signaling molecules and pathways that mediate
cell interactions during development. Previous experiments
involving the isolation, dissociation and recombination of

616 BioEssays 24.7


blastomeres have shown that ascidian embryos also utilize
complex, yet conserved, cell–cell communications to specify
early embryonic cell fates that are similar to those in other
organisms.(3) Developmental fates are specified by cell inter-
actions in mesodermal tissues such as notochord, mesench-
yme, secondary muscle and trunk lateral cells, in ectodermal
tissues such as the central and peripheral nervous systems,
and also in posterior endoderm. Thus, it is now realized that the
development of ascidian embryos is not entirely mosaic.
The importance of cell signaling in ascidian embryos is
supported by the results of treatment with an inhibitor of MEK/
MAPKK, which suppresses the formation of all of the tissues
listed above.(44) By contrast, the treatment does not affect the
formation of tissues whose fates are specified by maternal
determinants. MEK/MAPKK is a protein kinase involved in
various kinds of signal transduction by activating a mitogen-
activated protein kinase (MAPK, also known as extracellular
signal-regulated kinase, ERK).(45,46) Similarly, treatment with
an inhibitor of fibroblast growth factor receptor (FGFR) results
in loss of most of the above tissues, except for trunk lateral
cells and posterior endoderm. Therefore, an FGF and MEK-
MAPK signaling cascade is widely involved in embryonic
inductions in ascidians.
Notochord induction
Mesodermal tissues are derived from the marginal zone of
the vegetal hemisphere. Fig. 4A–C shows a fate map of the
vegetal hemisphere at the blastula stage (32- to 64-cell stage).
Cell lineages that give rise to notochord and mesenchyme
are shown in Fig. 4D,E. The notochord is one of the most
intensively analyzed tissues in ascidian embryos because it
is one of the hallmark morphological characteristics of any
chordate. In ascidians, 40 notochord cells are located in the
larval tail. 32 of them are called primary notochord cells and
the 8 cells situated in the caudal tip region are designated
secondary notochord cells. The primary notochord cells origi-
nate from four notochord precursor blastomeres (colored pink)
in the anterior marginal zone of the vegetal hemisphere of the
64-cell embryo. The most-remarkable feature of studies of
inductive cell interactions in ascidian embryos is that induc-
tion can be analyzed at the single cell level. Isolation and
recombination of presumptive notochord blastomeres have
revealed that inductive interactions mediate the determina-
tion of notochord fate.(47,48) As summarized in Table 1, this
induction occurs at the 32-cell stage and notochord pre-
cursors acquire developmental autonomy at the 64-cell stage.
Inducers of the primary notochord are the endoderm blas-
tomeres (colored yellow in Fig. 4). Notochord blastomeres of
32-cell embryos can also induce notochord fates in neighbor-
ing notochord blastomeres. Only presumptive notochord
blastomeres are competent and can respond to the specific
kinds of endodermal signals that induce them to differentiate
into notochord cells. Even in presumptive notochord blasto-
Review articles
meres, competence is lost at the 44-cell stage (just after the
cleavage of notochord blastomeres in the 32-cell embryo).
Fibroblast growth factor (FGF), unlike activin, is a signaling
molecule that mediates notochord induction.(49) Overexpres-
sion of the dominant negative form of the FGF receptor, or
treatment of embryos with a specific inhibitor of FGF receptor,
indicate that the inductive signal is received by the FGF
receptor.(44,50) FGF signaling is known in many animals to be
transduced within the cell by Ras–Raf–MEK–MAPK signal-
ing cascade. During ascidian notochord induction, Ras and
MEK are also required, and eventually MAPK is phosphory-
lated and activated.(44,51) Consequently, transcription of a
Brachyury homolog (HrBra, formerly As-T) becomes activated
in notochord cells at the 64-cell stage by an as-yet-unknown
mechanism.(49,52,53) In vertebrates, Bra is known to be a
transcription factor involved in mesoderm formation.(54) In
ascidians, Bra is expressed exclusively in notochord pre-
cursors. It plays a central role as a transcription factor in
the notochord formation process because injection of HrBra
mRNA into eggs promotes ectopic notochord cell forma-
tion,(55) and, in this case, isolated blastomeres are able to
differentiate autonomously into notochord without induction.
Recent study has shown that another transcription factor,
HNF-3, acts synergistically with HrBra during notochord
differentiation, similar to what has been observed in frog
embryos, although HNF-3 is expressed in most blastomeres of
the vegetal hemisphere before notochord induction starts.(56)
To identify the genes downstream from Brachyury,
subtractive hybridization screening was carried out using
Brachyury-overexpressing Ciona embryos. A total of 19 genes
were found to be notochord-specific and another 20 were
expressed predominantly in the notochord.(57–59) Expression
of these genes starts at various stages ranging from gastrula
to tailbud. At least one of them, a tropomyosin-like (Ci-trop)
gene, is reported to be a direct target of Brachyury.(60) This
gene has indispensable Ci-Bra-binding sites in its controlling
cis-element. Along with fossil and other information, these new
molecular findings will hopefully facilitate our continued quest
to better understand how developmental processes evolved
to generate the basic chordate body plan that is characterized
by formation of the notochord.
Mesenchyme induction
Two bilateral mesenchyme cell clusters are located between
the ventromedial endoderm and ventrolateral epidermis in the
trunk region of the larva (Fig. 4C). Mesenchyme cells exclusiv-
ely originate from four precursor blastomeres (colored green)
in the posterior-lateral marginal zone of the vegetal hemi-
sphere of the 64-cell embryo (Fig. 4B). Isolation and recombi-
nation of presumptive mesenchyme blastomeres was carried
out.(61) Striking similarities were found between the notochord
and mesenchyme inductive mechanisms, as summarized in
Table 1. The similarity is noticeable just by looking at the cell
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Figure 4. Diagrams showing fates of cells in the vegetal hemisphere of ascidian embryos. A–C: The name of each blastomere is
indicated. Endoderm (En)-lineage cells are colored yellow. Mesenchyme (Mes)-lineage cells are shown in green and muscle (Mus)-
lineage cells in red. Notochord (Not)- and nerve cord (NC)-lineage cells are colored pink and purple, respectively. A: 32-cell embryo.
Vegetal view. Anterior is up. B: 64-cell embryo. Blastomeres connected with a bar are sister blastomeres. C: Tailbud embryos. Lateral
views. Upper and lower diagrams illustrate midsagittal and parasaggital sections, respectively. D,E: Lineage trees in the vegetal
hemisphere. As development is bilaterally symmetrical, one side of the embryo is shown. D: Lineage tree relevant to the primary
notochord lineage and starting from the anterior-vegetal (A4.1) blastomere of the 8-cell embryo. E: Lineage tree starting from the
posterior-vegetal (B4.1) blastomere, from which mesenchyme and primary muscle cells originate. TVC, trunk ventral cells. (From Kim
GJ, Yamada A, Nishida H. Development 2000;127:2853–2862, with permission of The Company of Biologists Limited.(62))

lineage trees that generate notochord and mesenchyme
(Fig. 4D,E). Inductive interactions mediate the determination
of mesenchyme fate. This induction occurs at the 32-cell stage
and mesenchyme precursors acquire developmental autono-
my at the 64-cell stage. The inducer cells are endoderm
blastomeres. Only the presumptive mesenchyme blasto-
meres are competent and can respond to the endodermal
signal by differentiating into mesenchyme cells. Furthermore,
FGF, but not activin, is an important signaling molecule in this
process.(62) The signal is received by an FGF receptor(44,50)
and Ras and MEK appear to be required for intracellular
signaling.(44) Thus, the same signaling cascade down to MAPK
is utilized in both notochord and mesenchyme inductions.
Downstream transcription factors involved in this signaling
pathway, such as Bra in notochord induction, have not yet
been identified in mesenchyme induction. However, with
regard to gene expression, it is intriguing that the muscle
actin gene (HrMA4) is immediately downregulated after
induction. The expression of HrMA4 is precociously initiated
at the 32-cell stage in the muscle/mesenchyme (B6.2)
blastomeres (Fig. 4A) before fate restriction.(36) The B6.2
blastomere (mesenchyme/muscle precursor) divides into the
B7.3 (mesenchyme precursor) and B7.4 (muscle precursor)
blastomeres of the 64-cell embryo (Fig. 4B,E). The expression
continues only in the muscle blastomere, and is down-
regulated in the mesenchyme blastomere. The myosin heavy

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Table 1. Common features shared by notochord
and mesenchyme inductions
Cellular level
1. Their origins in the cell lineage tree show similar topology.
2. Inductive interactions are required.
3. The inductions start at the 32-cell stage before fate restriction.
4. The precursors acquire developmental autonomy at the 64-cell
stage.
5. Competence to the inductive signal is lost at least at the 64-cell
stage.
6. Endoderm blastomeres are the inducer.
7. Only the precursors have competence.
8. Only one daughter cell of the induced blastomeres assumes a
notochord or mesenchyme fate.
Molecular level
1. FGF is a potent inducer, but activin is not.
2. FGF receptor is required for induction.
3. Ras and MEK transduce the signal intracellularly.
chain gene is expressed in the same way. When the B6.2 cells
are isolated or whole embryos are treated with MEK inhibitor to
inhibit induction, actin expression continues in both daughter
cells, and eventually both of them develop into muscle cells.(61)
Therefore, inductive interactions cause the immediate down-
regulation of muscle-specific genes and suppress the muscle
fate of presumptive-mesenchyme blastomeres.
Suppression of the muscle fate of mesenchyme precursors
by cell interactions implies that the muscle determinant,
macho-1 protein, is also distributed in mesenchyme precur-
sors. When a moderate amount of macho-1 mRNA is injected
into eggs, ectopic formation of muscle occurs in non-muscle
blastomeres. Under these conditions, mesenchyme and noto-
chord precursors rarely transfate into muscle even if epidermis
and endoderm precursors develop into muscle (K. Sawada
and H. Nishida, unpublished data), although injection of a
high dose of the macho-1 mRNA can confer a muscle fate on
most embryonic cells, including mesenchyme and notochord
blastomeres. This observation suggests that an endodermal
signal may repress muscle fate by suppressing or modi-
fying macho-1 function, and that emission of the signal from
the vegetal blastomeres is executed independently of the
endoderm fate of vegetal pole blastomeres.
Responsiveness of signal-receiving
blastomeres
In the anterior marginal zone of the vegetal hemisphere,
primary notochord is induced in the area flanked by endoderm
and nerve cord blastomeres (Fig. 4). The ‘nerve cord’ desig-
nates the posterior neural tube located in the trunk and tail
region of the larva with the ‘brain’ vesicle anteriorly derived
from the animal hemisphere. In the posterior-lateral marginal
zone, mesenchyme is induced in the area flanked by endo-
derm and muscle blastomeres. As mentioned before, there
are striking similarities at the cellular and molecular levels
Review articles
between notochord and mesenchyme inductions (Table 1).
This implies that a similar mechanism symmetrically functions
in both the anterior and posterior marginal zones. What, then,
are the important mechanistic differences in the specifica-
tion processes that underlie notochord and mesenchyme
formation?
In normal embryos, the notochord is induced by anterior
(A-line) endoderm blastomeres, while mesenchyme is induced
by posterior (B-line) endoderm. First, we examined whether
the type of tissue induced depends on the inducing anterior
and posterior endoderm or on the responding blastomeres
by carrying out blastomere recombinations at the 32-cell
stage.(62) When an anterior endoderm blastomere was recom-
bined with a posterior mesenchyme precursor, mesenchyme
was formed, and when a posterior endoderm blastomere was
recombined with an anterior notochord precursor, notochord
was formed. These results supported the latter possibility, i.e.
that the type of tissue induced depends on the responding
blastomeres. There is no difference between the inducing
abilities of the anterior and posterior endoderm.
The results of FGF treatment also support this idea.(62)
Presumptive notochord blastomeres respond to this molecule
by forming notochord; mesenchyme is never formed. Simi-
larly, mesenchyme precursors are induced by FGF to form
mesenchyme, and not to form notochord. Thus, a single
signaling molecule promotes two types of response: the
formation of the notochord and that of the mesenchyme.
Therefore, presumptive mesenchyme and notochord blasto-
meres differ in their responsiveness.
What, then, brings about this difference in responsiveness?
Logically the difference must lie within the responding cells
themselves. One possibility is that egg cytoplasmic factors
differentially partitioned into each blastomere, determine the
difference. To examine this possibility, removal and trans-
plantation of egg cytoplasm was carried out by microsurgery.
Egg fragments containing posterior-vegetal cytoplasm (PVC)
were removed or transplanted to the anterior region of another
intact egg after completion of ooplasmic segregation. PVC is
the region corresponding to Conklin’s myoplasm at completion
of ooplasmic segregation, and macho-1 mRNA is localized
there (Fig. 2B, right panel). The experimental results are
shown schematically in Fig. 5. Removal of the PVC resulted in
anteriorization of the embryo. The blastomeres positioned
where mesenchyme blastomeres are normally located were
converted to notochord, so that central endoderm blastomeres
were encircled by notochord blastomeres.(62,63) Thus, removal
of the PVC causes ectopic formation of notochord and loss of
mesenchyme in the posterior region (Fig. 5B). By contrast,
removal of the anterior cytoplasm had no effect on embryo-
genesis, including notochord formation.
Transplantation of the PVC to the anterior region sup-
pressed notochord formation and promoted ectopic formation
of mesenchyme in the anterior blastomeres that is never
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Figure 5. The posterior-vegetal cytoplasm (PVC) factor modifies the responsiveness of the signal-receiving blastomeres. Blastomeres
are represented schematically by rectangles. For precise arrangement of blastomeres, see Fig. 4A,B. A: Normal embryos. In the
anterior region, notochord (Not) is induced, while in the posterior region, mesenchyme (Mes) is induced next to endoderm (En). NC,
nerve cord; Mus, Muscle. B: PVC-removed embryo. In the posterior region, notochord is induced in place of mesenchyme. C: PVC-
transplanted embryo. In the anterior region, mesenchyme is induced instead of notochord.

observed in normal embryos (Fig. 5C).(62,63) By contrast,
development was normal when the anterior cytoplasm was
transplanted to the posterior region. In conclusion, the factors
that are localized in the PVC seem to be involved in generating
differences in cell responsiveness. In the presence of PVC
factors, blastomeres respond to the endoderm signal by
forming mesenchyme and, in their absence, blastomeres
respond by developing into notochord. The results of removal
and transplantation of the anterior cytoplasm suggest that no
important factors are localized in the anterior region.
The molecular identity of the PVC factor is still unknown.
One obvious candidate is macho-1, because PVC is the region
where macho-1 mRNAs is localized and macho-1 protein
would also be present in mesenchyme blastomeres, as
discussed above. Alternatively, the function of the PVC factor
may be attributable to some of the type I postplasmic RNAs
that were found in the maternal cDNA project and show a
localization pattern similar to that of macho-1.(10,64) Down-
stream to the PVC factor, zygotic genes may work to suppress
the notochord fate. Snail is a possible candidate because it
is expressed in muscle and mesenchyme precursors at the
32-cell stage. Furthermore, Snail is a zinc-finger protein known
to be a transcription repressor. Misexpression of Snail in
notochord-lineage cells suppresses at least the expression of
reporter genes driven by the Brachyury minimal promoter.(65)
Therefore, even if MAPK is activated by the endodermal signal
in mesenchyme blastomeres, Snail might suppress expres-
sion of the Brachyury gene. The global picture of mesoderm
patterning in the marginal zone is now known. In contrast to the
situation in vertebrates,(66) ascidian mesodermal patterning
does not seem to involve a graded signal.
Directed signaling and asymmetric
cell divisions
Another conspicuous feature of notochord and mesenchyme
inductions is the asymmetric cell divisions of their progenitor
cells. Recent evidence suggests that these inductions occur
at the 32-cell stage. Experiments involving recombination of
isolated blastomeres at various stages, and experiments
determining the periods of sensitivity to FGF treatment and
sensitivity to both the FGF receptor inhibitor and the MEK
inhibitor, all support the idea that the inductive interactions
for notochord and mesenchyme formation are initiated at the
32-cell stage.
It is important to recall that, in the ascidian cell lineage tree,
the fates of responding blastomeres are not yet restricted
to formation of a single kind of tissue at the 32-cell stage
(Figs. 4, 6). Endoderm precursors lie in the center. In the
anterior region, notochord/nerve cord precursor blastomeres
of the 32-cell-stage embryo divide into notochord (pink) and
nerve cord (purple) precursors of the 64-cell-stage embryo.
Similarly in the posterior region, mesenchyme/muscle pre-
cursor blastomeres of the 32-cell-stage embryo divide into
mesenchyme (green) and muscle precursors (red) of the
64-cell-stage embryo. Therefore, the separation of each cell
fate occurs at the 64-cell stage after induction has taken place,
and only one daughter blastomere assumes the induced fate.
Induced notochord and mesenchyme blastomeres always
face the inducing endoderm at the 64-cell stage. Thus,

620 BioEssays 24.7

 Review articles

Figure 6. A directed signal and asymmetric division model of the tissue specification mechanism in the vegetal hemisphere of the
ascidian embryo. The model is applicable to both the anterior and posterior margins of the vegetal hemisphere. A: Vegetal view of
the 32-cell embryo showing endodermal FGF signal (arrows) and presence of the PVC factor (red oblique lines) in the posterior
blastomeres. B: Vegetal view of the 64-cell embryo after completion of inductions and asymmetric divisions in both the anterior and
posterior regions. C: (1) Schematic drawing representing embryo at the 32-cell stage. Endoderm precursors (En) emanate an inductive
FGF signal (arrows) to neighboring anterior and posterior blastomeres and polarize them. The PVC causes different response in the
posterior marginal cells. (2) Asymmetric divisions occur at the 64-cell stage. In the anterior region, one daughter cell that faces the
inducer and does not have the PVC assumes a notochord fate (Not). In the posterior region, one daughter cell that faces the inducer and
contains the PVC adopts a mesenchyme fate (Mes). (3) Without an inductive signal, both daughter blastomeres in the anterior region
assume the default nerve cord fate (NC), and those in the posterior region assume the default muscle fate (Mus). (4) When isolated
blastomeres receive the FGF signal over their entire surface, both daughter cells develop into notochord or mesenchyme, depending on
the absence or presence of PVC. From Minokawa T, Yagi K, Makabe KW, Nishida H. Development 2001;128:2007–2017, with
permission of The Company of Biologists Limited.(67))

notochord and mesenchyme induction occurs such that only
one of the daughters of the induced blastomere in the 32-cell
embryo adopts a notochord or mesenchyme fate.
As described previously, mesenchyme blastomeres devel-
op into muscle when they do not receive an inductive signal.
Therefore, muscle is a default fate of muscle/mesenchyme
precursors. It has been reported that nerve cord is a default
fate of notochord/nerve cord precursors (Fig. 6C).(67) When
notochord and nerve cord precursors are isolated at the 64-cell
stage after completion of induction, and further cell division is
inhibited by cytochalasin B, the notochord blastomere even-
tually expresses a notochord differentiation marker, Not-1
antigen, while the nerve cord blastomere expresses the neural
plate marker genes HrETR-1 and HrTBB2 that encode RNA-

BioEssays 24.7 621

Review articles
binding protein(68) and b-tubulin,(69) respectively. By contrast,
when notochord/nerve cord precursors are isolated at the
32-cell stage and allowed to divide once, then further cleav-
ages are arrested, both blastomeres express neural plate
markers, and not notochord marker. Treatment with inhibitors
of FGFR and MEK also causes both daughter cells to assume
the nerve cord fate. The autonomy of nerve cord fate speci-
fication was also confirmed by isolation of precursor blasto-
meres at various stages and by dissociation of embryonic
cells. This was a surprise because the nerve cord of ascidian
larvae is formed by neural tube closure, as in vertebrates.
These findings, using HrETR-1 and HrTBB2 as molecular
markers, do not tell us whether the morphogenetic processes
of neural tube formation in ascidian larvae require cell inter-
actions. However, they do indicate that the initial step of nerve
cord specification during cleavage is autonomous.
Treatment with FGF gives opposite results. FGF treatment
of mesenchyme/muscle precursors at the 32-cell stage
causes both of the daughter blastomeres to develop into
mesenchyme.(62) Similar treatment of notochord/nerve cord
precursors confers a notochord fate on both daughter cells.(67)
Therefore, in the asymmetric divisions that occur in the anterior
region, nerve cord appears to be a default cell fate and
notochord is an induced fate; in the posterior region, muscle is
the default fate and mesenchyme is the induced fate.
Figure 6 summarizes our model. In normal embryos, the
precursor cells receive an endoderm signal from the vegetal
pole, and only one of the daughter cells facing the endoderm
assumes an induced cell fate. Directed signals that emanate
from endoderm blastomeres may polarize the responding
blastomeres at the 32-cell stage and promote asymmetric
divisions that operate in both the anterior and posterior regions.
Presumably, FGF signaling causes localized changes in the
mother cell. Then one of the daughter cells that faces the
endoderm is fated to notochord or mesenchyme depending on
the presence or absence of the PVC factors. This directed
signaling and asymmetric division model is supported by the
fact that treatment of isolated blastomeres with FGF in
seawater causes both daughters to assume a mesenchyme
or a notochord fate, because isolated mother blastomeres
receive the signal over the entire cell surface.
Similar examples can be found in the C. elegans embryo. At
the 4-cell stage, the EMS blastomere receives inductive
signals from the posterior P2 blastomere. Then it divides
asymmetrically into the anterior MS cell (muscle, neuron,
somatic gonad precursor; default fate) and posterior E cell
(gut precursor; induced fate).(70–72) In this case, the signaling
molecule is Wnt.(73) Moreover, it has been suggested that Wnt
signaling may be globally involved in binary fate specifications
that are accompanied by asymmetric cell divisions along the
entire anterior-posterior axis in later embryogenesis.(74–76)
Thus, directed-signal-mediated asymmetric divisions appear
to be widely utilized as a mechanism to generate cell fate
622 BioEssays 24.7
diversity in early embryos as well as later development in
various kinds of animal.
Future directions
We can now begin to understand how developmental fates are
determined at the cellular and molecular levels in most cell
types in ascidian embryos. While our molecular knowledge is
still incomplete, we have now characterized many of the likely
key molecules involved in autonomous fate specification
(localized RNAs) and cell interactions (signaling molecules).
Chasing the localized maternal factor has revealed macho-
1 as a muscle determinant whose presence had been
predicted a century ago. Similar to Drosophila and Xenopus
embryos,(77–79) the localized determinant in ascidian eggs is
maternal mRNA that encodes a transcription factor. So far, it
seems common that localized egg mRNA plays critical roles
in the initial steps of early embryogenesis. However, any
conclusion should be made with care, because the isolation of
many important localized RNAs may be technically simpler
than finding localized proteins. Ascidian would provide a good
system to analyze the mechanisms how specific mRNAs
are localized and relocated in eggs and embryonic cells.(80)
In order to understand the muscle-forming cascade, it will be
important to analyze epistatic relationships between muscle-
specific genes such as Tbx6(37,38) and the myogenic
factor(39,40) genes that are known to be expressed zygotically
in ascidians. A search for macho-1 homologs in other orga-
nisms should also be made.
We have learned much from analyses of notochord and
mesenchyme inductions, and a simple model of binary speci-
fication of cell fates operating in the marginal zone of the
vegetal hemisphere in ascidian embryos has been proposed.
The first step depends on the presence or absence of PVC
factors, and the second step is regulated by the presence or
absence of inductive interactions. From a comparative view-
point, it is remarkable that ascidians utilize cellular and
molecular mechanisms to pattern mesodermal tissues that
are significantly different from those in vertebrates. Ascidians
seem to have adopted a ‘‘digital’’ approach, rather than the
‘‘analog’’ one in vertebrates where there is graded activity of
BMP signaling.(66) This difference is probably related to the
fact that ascidian embryos consist of relatively few cells, and
that restriction of developmental fates occurs at a very early
stage. Another reason may be egg sizes. There may not be
enough distance between cells to generate graded activity of
signaling. Nevertheless, it is amazing that both phylogeneti-
cally related animals can show a similar fate map and generate
a similar basic body plan using substantially different mecha-
nisms. One explanation is that the pressure of natural selection
to conserve a body plan is high, but may not be so severe as to
restrict the way in which the ideal body plan is attained.
In induced asymmetric divisions, extracellular signaling
molecules and intrinsic factors are thought to combine in a

progenitor cell before it divides, conferring particular fates on
the two progeny cells. Intrinsic factors play roles in defining the
‘responsiveness’ or ‘competence’ of the progenitor cells. In
future studies of ascidian embryogenesis, identification of the
PVC factor and analysis of the intracellular events involved in
asymmetric divisions will be important.
We observed that each blastomere shows a distinct
response to the same signal, although the signaling cascade
is markedly conserved among each blastomere in ascidians,
as well as in other organisms that have been studied.
Therefore, it will be important to understand the mechanisms
that determine the way in which different blastomeres res-
pond differently. Clarification of maternal factors will no doubt
contribute to this understanding, as has been the case in
notochord and mesenchyme inductions. The problem is how
cells integrate the intrinsic activity of the PVC factor with the
information from extrinsic cues that are delivered into the cell
by the signal-transduction machinery.
Directed-signal-mediated asymmetric divisions play crucial
roles in the generation of cell diversity in ascidian embryos. It is
still unclear how mother cells are polarized before asymmetric
divisions, and cytological investigation of intracellular events
will be required to answer this question. Understanding of the
spatial details of activation of the MAPK pathways in signal
transduction will provide clues for the study of asymmetric
divisions in ascidian embryos.
Acknowledgment
I am grateful to the members of our HFSP group for helpful
discussions.
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