Patterning the marginal zone ofearly ascidian embryos: localizedmaternal mRNA and inductiveinteractionsHiroki Nishida

SummaryEarly animal embryos are patterned by localized eggcytoplasmic factors and cell interactions. In invertebratechordate ascidians, larval tail muscle originates from theposterior marginal zone of the early embryo. It hasrecently been demonstrated that maternal macho-1mRNA encoding transcription factor acts as a localizedmuscle determinant. Other mesodermal tissues such asnotochord and mesenchyme are also derived from thevegetal marginal zone. In contrast, formation of thesetissues requires induction from endoderm precursors atthe 32-cell stage. FGF–Ras–MAPK signaling is involvedin the induction of both tissues. The responsiveness forinduction to notochord or mesenchyme depends on theinheritance of localized egg cytoplasmic factors. Pre-vious studies also point to critical roles of directedsignaling in polarization of induced cells and in subse-quent asymmetric divisions resulting in the formationof two daughter cells with distinct fates. One cell ado-pts an induced fate, while the other assumes a defaultfate. A simple model of mesoderm patterning in asci-dian embryos is proposed in comparison with that ofvertebrates. BioEssays 24:613–624, 2002.� 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

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 showcell-autonomous developmentwhich 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 ofmesodermal 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 fatemap (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

BioEssays 24:613–624, � 2002 Wiley Periodicals, Inc. BioEssays 24.7 613

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

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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 thenmove 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 domainenriched inmitochondria, 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 localizedmaternal determinants ofmuscle formation in the

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.

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614 BioEssays 24.7

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 bymonitoring 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 ofmuscle 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 inmacho-1-

depleted embryos indicated that only primary muscle cells

were lost (Fig. 3E,F). Injection of synthetic macho-1 mRNA

intomacho-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 transferredto the ventral side of the intact egg, a secondary dorsal axis is

still induced.(32) This observation indicates that b-cateninfunctions 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

Figure 2. Distribution of cytoplasmic determinants and macho-1 maternal mRNA in eggs. A: Distribution of muscle determinant duringooplasmic 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 maternalmacho-1mRNA 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)

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BioEssays 24.7 615

posterior-vegetal cytoplasm of the fertilized egg has the ability

to promote muscle formation when transferred into epidermis

blastomeres. By contrast, the cytoplasm ofmacho-1-depleted

eggs did not promote ectopic muscle formation in epidermis

blastomeres. Thus maternalmacho-1mRNA 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 fiveCCHH-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) andmyogenic factor.(39,40) Inascidians, theexpres-

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

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: Precursorblastomere 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 primarymuscle 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)

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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,

andalso 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-

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, formerlyAs-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, althoughHNF-3 is expressed inmost blastomeresof

the vegetal hemisphere before notochord induction starts.(56)

To identify the genes downstream from Brachyury,

subtractive hybridization screening was carried out using

Brachyury-overexpressingCionaembryos. 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. Alongwith fossil andother information, thesenew

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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BioEssays 24.7 617

lineage trees that generate notochord and mesenchyme

(Fig. 4D,E). Inductive interactions mediate the determination

ofmesenchyme 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, thesamesignalingcascadedown toMAPK

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

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 theposterior-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))

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618 BioEssays 24.7

chain gene is expressed in the sameway.When the B6.2 cells

are isolated orwhole embryos are treatedwithMEK inhibitor to

inhibit induction, actin expression continues in both daughter

cells, andeventually both of themdevelop intomuscle 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 themuscle fate ofmesenchyme precursors

by cell interactions implies that the muscle determinant,

macho-1 protein, is also distributed in mesenchyme precur-

sors. When a moderate amount ofmacho-1mRNA is injected

into eggs, ectopic formation of muscle occurs in non-muscle

blastomeres. Under these conditions, mesenchyme and noto-

chord precursors rarely transfate intomuscle even if epidermis

and endoderm precursors develop into muscle (K. Sawada

and H. Nishida, unpublished data), although injection of a

high dose of themacho-1mRNA 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

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) endodermblastomeres,whilemesenchyme 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 endodermblastomerewas 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 toConklin’smyoplasmat 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 endodermblastomeres

wereencircledbynotochordblastomeres.(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

Table 1. Common features shared by notochord

and mesenchyme inductions

Cellular level1. 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 level1. FGF is a potent inducer, but activin is not.

2. FGF receptor is required for induction.

3. Ras and MEK transduce the signal intracellularly.

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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 PVCseem 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.

Oneobvious candidate ismacho-1, becausePVC 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 genesmaywork 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 ifMAPK 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 themarginal zone is nowknown. 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,

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.

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620 BioEssays 24.7

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 andnerve cordprecursors are isolated at the64-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-

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 andposterior 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 theinducer 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 onthe 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))

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BioEssays 24.7 621

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 andMEK 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 theasymmetric divisions that occur in theanterior

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 thatoperate inboth theanterior andposterior 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 ormesenchyme 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 canbe found in theC. elegansembryo. 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 isWnt.(73) Moreover, it has been suggested thatWnt

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

diversity in early embryos as well as later development in

various kinds of animal.

Future directions

Wecan nowbegin to understand howdevelopmental 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 localizedmaternal factor has revealedmacho-

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 bemade 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 showasimilar fatemapandgenerate

a similar basic body plan using substantially different mecha-

nisms.Oneexplanation is that thepressureof natural selection

to conserve a body plan is high, butmay 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

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622 BioEssays 24.7

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-mediatedasymmetric divisions play crucial

roles in the generation of cell diversity in ascidian embryos. It is

still unclear howmother 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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