Develop. Growth Differ. (1999) 41, 283–291

Changes in the adhesive properties of dissociatedand reaggregated Xenopus laevis embryo cells
Hiroki Kuroda,1 Hirofumi Sakumoto,1 Kei Kinoshita2 andMakoto Asashima1,2*
1

Department of Life Sciences and 2CREST Project, Department of Life Sciences, Graduate School ofArts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.

Activin A is a member of the transforming growth factor b superfamily, and the strongest candidate mesoderminducer. The initial adhesive property changes in amphibians are likely to be mediated by mesoderm-inducers
like activin A. The manner in which these changes actually occur, however, remains poorly understood. In thepresent study, the adhesive property changes mediated by activin A were directly demonstrated. Activin A
functioned as a morphogen at low concentrations (less than 0.5 ng/mL), with no effect on the type A adhesive
property. But at high concentrations (1 ng/mL), it induced another type of adhesive property, type N, and atvery high concentrations (more than 10 ng/mL), it induced yet another type of adhesive property, type Y. Cells
that have types A, N, and Y adhesive properties ultimately differentiated into atypical epidermis, notochord,and yolk-rich cells, respectively. It was also shown that these changes occurred between 5 and 10 h after
induction by activin A. The implications of these results for the relationship between the adhesive property
acquired during early and later stages of differentiation are also discussed.
Key words:

activin A, adhesive property, animal cap, mesoderm-inducer, Xenopus laevis.

Introduction
Regulated changes in cell adhesion play an importantrole in establishing the embryonic body plan duringvertebrate development. The initial changes in embryonic cell adhesion during amphibian development may
be regulated by mesoderm-inducers, but these putative signals have not been isolated to date. One candidate molecule for these primary events is activin A,
a member of the transforming growth factor (TGF)-b
superfamily. Activin A has very potent mesoderminducing activity on Xenopus presumptive ectoderm
and induces various mesodermal tissues in a dosedependent manner. Presumptive ectoderm treated withactivin A differentiates into ventral mesodermal tissue
such as coelomic epithelium at low activin A concentrations, muscle at high concentrations, and thedorsalmost tissue, notochord, at very high concentrations (Asashima et al. 1990; Ariizumi et al. 1991a,b). In
addition, cells dissociated from animal caps haveseveral prospective cell fates dependent on distinct
activin A concentration thresholds (Green et al. 1994).

*Author to whom all correspondence should be addressed.
E-mail: cmasa@komaba.ecc.u-tokyo.ac.jp
Received 11 September 1998; revised 30 November 1998;
accepted 22 December 1998.

In explantation experiments, ectoderm dissectedfrom late morula–early gastrula stage embryos treatedwith a high concentration of activin A (100 ng/mL)
formed only yolk-rich endodermal cells. When the
explants were transplanted into the blastocele of otherearly gastrula, they formed part of the endoderm of thehost embryo and induced a secondary axis with only
posterior characteristics, including axial mesoderm andneural tissues. In contrast, whole secondary axes were
induced when activin-treated ectoderm was transplanted into the ventral marginal zone (VMZ) of earlyblastula; the transplanted pieces invaginated anddifferentiated into foregut structures, including thepharynx, stomach, and liver. These phenomena werealso observed in experiments in which presumptive
foregut was used instead of activin-treated ectoderm.
These findings show that activin-treated ectoderm
undergoes changes in adhesive properties and can act
as the single organizing center in Cynops (Ariizumi &Asashima 1995; Ninomiya et al. 1998). In addition,
when presumptive epidermal explants from pigmented
neurula embryos and neural plate explants from unpigmented neurula embryos were dissociated and mixed,
the cells reaggregated such that epidermal cells
covered other cell types (Townes & Holtfreter 1955).
In the present study, we used a new in vitro assay
to determine how changes in cell–cell adhesion
occur in the early stages of development and directly

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demonstrated that cells induced by activin A undergochanges in adhesive properties and subsequently
differentiate.

Materials and Methods

Embryos and activin A solution
Xenopus laevis eggs were obtained from females byinjecting them with 400 units of human chorionicgonadotropin (Gestron; Denka Seiyaku Co., Kawasaki,
Japan), fertilized in vitro with minced testis, andcultured in 1 3 Steinberg’s solution (58 mM sodium
chloride, 0.67 mM potassium chloride, 0.34 mM calcium
nitrate, 0.83 mM magnesium sulfate, 100 mg/L kanamycin sulfate and 4.6 mM timza base, pH 7.4). Staging
was according to Nieuwkoop and Faber (1967). The
jelly coat was removed with 3% cysteine hydrochloride
in Steinberg’s solution (pH 7.8), and the vitelline
membrane was manually removed with fine forceps. All
operations were carried out under sterile conditions.
Human recombinant activin A (provided by Dr
Yuzuru Eto, Ajinomoto Co. Inc., Kawasaki, Japan) was
dissolved at concentrations of 0.05–100 ng/mL in 1 3
Steinberg’s solution containing 0.1% bovine serum
albumin (BSA; A-7999; Sigma Chemical Co., St Louis,
MO, USA).

gation at 100 g and cultured for 5, 10, 20 h or 3 days.
(Fig. 1).
The reaggregates were fixed in phosphate-buffered
10% commercial formalin for 5 h. They were then dehydrated through an ethanol series, embedded in paraffin, and sectioned at 6 µm. After deparaffinization,
the sections were mounted in PermaFluor Aqueous
Mounting Medium (434990; Immunon, Pittsburgh,
USA) and examined under an epifluorescence microscope.

Histological examination
Reaggregates were fixed with Bouin’s fluid for 3 h,dehydrated with ethanol, embedded in paraffin wax,
sectioned at 6 µm and stained with hematoxylin andeosin (HE).

Results

Activin A has more potent and distinct effects on
dissociated cells than on intact animal caps
Activin A dose-dependently induces animal caps toform several different types of cell, with higher doses
eliciting more dorsal tissues (Asashima et al. 1990;
Ariizumi et al. 1991a,b). Preliminary experiments were

Reaggregation experiment
Embryos at the 2-cell stage were injected with a total
volume of 12 nL of 1% Texas red-dextran-amine (TRDA;
D-1863; Molecular Probes, Eugene, OR, USA) or 1%
fluorescein-dextran-amine (FDA, D-1820; Molecular
Probes).
The 0.4 3 0.4 mm X. laevis presumptive ectoderm
was cut from stage 8.5 embryos in 3% agar-coatedculture dishes (35 mm Tissue Culture Dish, 3000–035;
Iwaki Glass, Chiba, Japan) containing calciummagnesium-free Dulbecco’s phosphate-buffered saline(CMF-PBS, D-5652; Sigma) using tungsten needles.
The presumptive ectodermal regions were transferred to another 3%-agar-coated culture dish filled with
CMF-PBS. The outer layer of cells, which is difficult todissociate, was discarded, and the inner layer was
disaggregated into a single-cell suspension by gentle
pipetting.
Cells from 10 caps were incubated in 5 mL diluted
activin A solution with Steinberg’s solution containing
0.1% BSA. After 1 h, the cells were swirled into thecenter of the dish, the activin A solution was replacedwith fresh Steinberg’s solution, and the cells were gently
resuspended. This operation was performed threetimes. Reaggregates were prepared by gentle swirling
until initiation of reaggregation or by gentle centrifu-

Fig. 1. Schematic diagram of the experimental procedures.
Xenopus laevis embryos at the 2-cell stage were injected with a
total volume of 12 nL of 1% Texas red-dextran-amine (TRDA,
shown in red), or with a total volume of 12 nL of 1% fluoresceindextran-amine (FDA, shown in green). The embryos were grown
to stage 8.5, and a 0.4 3 0.4 mm segment of the presumptive
ectoderm (called animal caps) was then cut from the embryos.
The animal caps were then transferred to dishes filled withcalcium-magnesium-free solution. The outer layer of the animal
cap, which is difficult to dissociate, was discarded and the inner
layer was disaggregated into a single-cell suspension by
gentle pipetting. Dissociated cells derived from five caps andlabeled with TRDA or FDA were incubated in 5 mL of medium
with various concentrations of activin A: 0, 0.5, 1, 10, or
100 ng/mL. After 1-h incubation each dish of cells was washed,
and the cells were reaggregated by centrifugation at 100 g,
washed again, and incubated for 5, 10, 20 h or 3 days.

Adhesive properties of early embryo

performed to establish the overall dose-response of
dissociated animal cap cells to form differentiatedtissue. The effect of different activin A concentrations
on the adhesive properties of dissociated Xenopus
animal cap cells was examined (Fig. 2). The results
suggested a simple model for the effects of activin A

285

on dissociated cells. Low concentrations of activin A,
less than 0.5 ng/mL, induced only atypical epidermis(Fig. 2A–E). High concentrations, 0.75–5 ng/mL,
induced mostly notochord (Fig. 2F–I), and in particular,
1 ng/mL induced only notochord. Very high concentrations, exceeding 7.5 ng/mL, induced only yolk-richFig. 2. Effects of activin A on dissociated animal cap cells. Reaggregates treated with 0 (A), 0.05 (B), 0.1 (C), 0.25 (D), or 0.5 ng/mL
activin A (E) differentiated into atypical epidermis. Reaggregates treated with 0.75 ng/mL (F) activin A differentiated into notochord,muscle, and atypical epidermis. Reaggregates treated with 1 ng/mL (G) activin A differentiated only into notochord. Reaggregatestreated with 2.5 (H) or 5 ng/mL (I) differentiated into notochord and yolk-rich tissue. Reaggregates treated with 7.5 (J), 10 (K) or
100 ng/mL (L) differentiated only into yolk-rich tissue. mus, muscle; not, notochord; yr, yolk-rich tissue. Bars, 100 µm.

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tissue (Fig. 2J–L). These results indicate that activin A
has only two functions, induction of notochord andinduction of yolk-rich tissue. Thus, the effect of activin
A on the adhesive properties of Xenopus animal cap
cells was examined at concentrations of 0.5, 1, 10, and
100 ng/mL.

‘Sorting by separation’ occurs in reaggregatedmixed animal cap cells induced to form atypicalepidermis or yolk-rich tissues
Cells treated with 10 or 100 ng/mL activin A to induce
yolk-rich tissue were labeled with TRDA, and cells
treated with 0.5 ng/mL activin A and untreated cells,
which form atypical epidermis, were labeled with FDA.
Labeled cells were mixed, and reaggregates were incubated until control embryos had reached stage 38.
Reaggregates at this stage showed clear separationof TRDA-labeled and FDA-labeled cells (Fig. 3; Table 1).
For convenience, we refer to this type of sorting as‘sorting by separation’. Stained sections of fixed reaggregates showed that TRDA-labeled cells had differentiated into yolk-rich tissue and FDA-labeled cells intoatypical epidermis (data not shown).

‘Inner–outer sorting’ occurs in reaggregated mixed
animal cap cells induced to form notochord orother tissues

Fig. 3. ‘Sorting by separation’. Reaggregates formed by treating cells with activin A concentrations that induce yolk-rich tissue
and concentrations that induce atypical epidermis underwent‘sorting by separation’. (A) Reaggregates of cells treated with 0(green cells) or 100 ng/mL (red cells) activin A. (B) Reaggregatesof cells treated with 0.5 (green cells) or 100 ng/mL (red cells)
activin A. (C) Reaggregates of cells treated with 0 (green cells)or 10 ng/mL (red cells) activin A. (D) Reaggregates of cells
treated with 0.5 (green cells) or 10 ng/mL (red cells) activin A.

Cells induced by 10 ng/mL activin A to form yolk-richtissue were labeled with TRDA, and cells treated with
1 ng/mL activin A to form only notochord were labeledwith FDA. In reaggregates composed of the mixture ofthese cells, the FDA-labeled cells were located in acentral mass, surrounded by TRDA-labeled cells. We
referred to this type of sorting as ‘inner–outer sorting’(Fig. 4A,B; Table 1). Fixing and staining of reaggregatesconfirmed that the TRDA-labeled cells had differentiated into yolk-rich tissue and the FDA-labeled cells into
notochord (data not shown).
Table 1. Cell-lineage
reaggregatesFig. 4. ‘Inner–outer sorting’. Reaggregates formed from cells
treated with activin A concentrations that induce notochord, or
yolk-rich tissue or atypical epidermis, underwent ‘inner–outersorting’. (A) Reaggregates of cells treated with 1 (green cells)or 100 ng/mL (red cells) activin A. (B) Reaggregates of cells
treated with 1 (green cells) or 10 ng/mL (red cells) activin A. (C)Reaggregates of cells treated with 0.5 (green cells) or 1 ng/mL(red cells) activin A. (D) Reaggregates of cells treated with 0(green cells) or 1 ng/mL (red cells) activin A.

analysisofthecompositionofActivin concentration (ng/mL)
TRDA
FDA
Total
labeledlabeled
cells
cells

Number
IOS
SBS

NS

100
100
100
100
100
10
10
10
10
1
1
1
0.5
0.5
0

0
0
7
0
0
0
5
0
0
0
5
8
0
0
0

5
5
0
0
0
5
0
0
0
5
0
0
5
5
9

100
10
1
0.5
0
10
1
0.5
0
1
0.5
0
0.5
0
0

5
5
7
5
11
5
5
5
8
5
5
8
5
5
9

0
0
0
5
11
0
0
5
8
0
0
0
0
0
0

IOS, Inner–outer sorting; SBS, sorting by separation; NS, notsorting.

Adhesive properties of early embryo

In a second experiment, cells induced by 1 ng/mL
activin A to form only notochord were labeled with
TRDA, and another part of cells treated with 0.5 ng/mL
activin A or untreated, which differentiated into only
atypical epidermis, were labeled with FDA. In this case,‘inner–outer sorting’ occurred in reaggregates resulting
from mixing of these cells, and the TRDA-labeled cells
were located in a central mass, surrounded by FDAlabeled cells (Fig. 4C,D; Table 1). Histological examination confirmed that the TRDA-labeled cells had
formed notochord and the FDA-labeled cells atypicalepidermis (data not shown). In addition, although it isusually difficult to obtain histological information fromfluorescent sections, we had no difficulty detecting thevacuolation characteristics of notochord differentiationin these preparations.

Sorting does not occur in reaggregates preparedby mixing histologically identical cells
The TRDA-labeled and FDA-labeled cells in reaggregates did not sort when they had been treated withyolk-inducing tissue concentrations of activin A (10or 100 ng/mL), (Fig. 5A–C; Table 1), the notochordinducing concentration of activin A (1 ng/mL), (Fig. 5D;
Table 1), or the atypical epidermis-inducing concentration of activin A (0.5 ng/mL), or when they wereuntreated (Fig. 5E–G; Table 1). Regions in which red
and green cells were well mixed are shown as yellow
in Fig. 5. These results refute the hypothesis that different concentrations (here 0 and 0.5 ng/mL, and 10 and

287

100 ng/mL) result in formation of the same tissue, but
induce different adhesive properties. Rather, theseresults indicate that yolk-rich cells and notochord cells
induced by activin A, as well as atypical epidermal cells
unexposed to activin A, have a single type of adhesive
property.

Adhesive properties of animal cap cells maychange 5–10 h after induction mediated bymesoderm-inducers
When do changes in the adhesive properties of cells
induced by a mesoderm-inducer occur in vivo? To
answer this question, we examined the time course ofreaggregate formation (from initiation of regeneration,
5, 10, 20 h, and 3 days after induction with activin A).
There are two general methods to gather dissociatedcells. One is the centrifugation method, which we predominantly employed, and the other is the swirling
method, which we used to gather cells immediately
upon initiation of regeneration (Fig. 6D,I,N). Sorting
did not occur in reaggregates between the initiationof regeneration (Fig. 6D,I,N) and 5 h (Fig. 6E,J,O).
However, by 10 h, sorting was evident (Fig. 6F,K,P), andwas sustained through 20 h (Fig. 6G,L,Q) up to 3 days
(Fig. 6H,M,R). In control experiments, no sorting
occurred up to 3 days (Fig. 6A–C). These results
indicate that adhesive property changes occur 5–10 hafter induction by mesoderm-inducers, at the gastrula
stage. This is the first study to directly demonstrate thatadhesive property changes occur at this stage.

Fig. 5. Sorting did not occur when reaggregates were composed of two cell types having the same fate. (A) Reaggregates in which
both red and green cells were treated with 100 ng/mL activin A. (B) Reaggregates of cells treated with 10 (green cells) or 100 ng/mL(red cells) activin A. (C) Reaggregates in which both red and green cells were treated with 10 ng/mL. (D) Reaggregates in which
both red and green cells were treated with 1 ng/mL. (E) Reaggregates in which both red and green cells were treated with 0.5 ng/mL.
(F) Reaggregates of cells untreated (green cells) or treated with 0.5 ng/mL (red cells) activin A. (G) Reaggregates in which both red
and green cells were untreated with 0 ng/mL activin A (red cells). Yellow regions in Fig. 5 indicate the region of mixed red and green
cells.

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Fig. 6. Time course ofreaggregate formation. All
panels are sections ofreaggregates. (A–C) Reaggregates of untreatedcells. (D–H) Reaggregatesof cells treated with
100 ng/mL (red cells) and
0 ng/mL activin A (green
cells). (I–M) Reaggregatesof cells treated with
100 ng/mL (red cells) and1 ng/mL activin A (green
cells). (N–R) Reaggregates of cells treated with
1 ng/mL (red cells) and
0 ng/mL activin A (green
cells). (A,D,I,N) Immediately upon initiation ofreaggregation (≈ 2–3 h);(B,E,J,O) 5 h after reaggregation; (F,K,P) 10 h after
reaggregation; (G,L,Q)
20 h after reaggregation;(C,H,M,R) 3 days after
reaggregation.

Adhesive properties of early embryo

289

Discussion

Effects of activin A on dissociated cells

The finding that mesoderm-inducers change adhesive
properties in amphibians was predicted, but the mechanism underlying this phenomenon remains to be elucidated. As there is no apparent difference between thecells induced or not induced by mesoderm-inducers,
it is difficult to demonstrate these changes. In the present study, we used activin A, which is widely regarded
as the most promising candidate mesoderm-inducer,
plus fluorescent molecules and dissociated cells, anddirectly demonstrated that activin A changes adhesive
properties of blastomeres. We also showed that activin
A exerts this effect dose dependently and that thechange occurs 5–10 h after activin A treatment. These
results imply that mesoderm-inducers change celladhesive properties at the gastrula stage.

Activin A induces animal caps to form several different
types of cells in a dose-dependent manner. These are
ventral mesoderm in the form of coelomic epithelium,
blood corpuscles, and mesenchyme at very low concentrations (0.1 ng/mL), neural tissue and muscle at alow concentration (1 ng/mL), dorsal mesoderm in theform of notochord at a high concentration (10 ng/mL),and endoderm in the form of yolk-rich tissue at a very
high concentration (100 ng/mL). In dissociated cells,
however, we have detected only two tissue types: notochord and yolk-rich tissue. While we also recognizedsome muscle, it may have formed secondary to notochord induction. Why are other tissues not induced,
and why is the effect of activin A on dissociated cells
much stronger than on animal caps? We propose two
hypotheses. One is that dissociated cells have more
receptors and bind activin A more readily than animal
cap cells, and each dissociated cell has an almost
equal number of activin A receptors, making theinduced tissue nearly homogeneous. The second
is that reaggregates may no longer possess themolecules to function competitively or cooperatively
with activin A. The most probable candidate for thecompetitive molecule is bone morphogenetic protein4 (BMP-4) (Dale et al. 1992; Jones et al. 1992; Fainsod
et al. 1994). The BMP-4 is a secreted protein that binds
to the dorsalization and neuralization inducer proteins
chordin and noggin (Piccolo et al. 1996; Zimmerman
et al. 1996), which are induced by activin A. Thus, it
seems reasonable to assume that these proteins are
removed by dissociation, thereby enhancing activin A
activity. The most probable candidate for the cooperative molecule is fibroblast growth factor (FGF). The
FGF is a secreted protein, and it is a highly effective
inducer of mesoderm, equivalent to activin A. We propose that activin does not have multiple functions as
a morphogen, as previously thought, but that its solefunction is simply to induce notochord or endoderm,and muscle may be induced cooperatively by activin
A and FGF. The many types of tissue induced by treating the animal cap with activin A may result from induction by other cofactors, or from secondary inductionof another cell type induced by activin A or other cofactors.

Two types of adhesive properties are induced by
activin A
Reaggregates composed of untreated animal cap cells
or of cells treated with less than 0.5 ng/mL activin A differentiate into atypical epidermis, while reaggregatescomposed of cells treated with 0.75–5 ng/mL activin A
differentiate into notochord. Cells treated with more than
7.5 ng/mL activin A differentiate into yolk-rich cells. In
addition, we found that these three cell types havespecific and distinct adhesive properties. The adhesive
properties of the cells treated with less than 0.5 ng/mL,with 0.75–5 ng/mL, and with more than 7.5 ng/mL ofactivin A are, for convenience, referred to as type A(atypical epidermis), type N (notochord), and type Y(yolk-rich tissue), respectively. In other words, activin
A induces animal cap cells to dose-dependently manifest distinct adhesive properties, with low, high, andvery high doses eliciting types A, N, and Y, respectively.
According to the thermodynamic model of cell–cellinteraction (Steinberg 1964), the binding activity of cellswith type N adhesion appears to be very strong,
because the cells move into the central region of areaggregate whenever it is composed of cells with type
A or Y adhesion. This is an important concept forunderstanding the formation of the notochord in vivo,
as discussed below. The binding activity of cells with
type Y adhesion, however, may be weak because reaggregates composed of cells treated with a high concentration of activin A differentiate into yolk-rich tissue
and become smaller and the reaggregate ultimately
disintegrates (data not shown). In light of these results
and the thermodynamic model (Steinberg 1964), we
advocate the former hypothesis (expressing thebinding activity between cells having type A and type
N adhesivity as ‘S (a-n)’, where:
S (n-n) > S (a–n) 7 S (n–y) ≥ S (a–a) > S (y–y) ≥ S (a–y)

Mechanism of notochord formation in vivo
The results of the present study suggest the following
model for the mechanism of early development of
X. laevis. Cells from the Nieuwkoop center region involute along with the dorsal mesoderm during gastrulationand serve as a continuing source of an activin A-likesignal resulting in generation of notochord and otherstructures (Vodicka & Gerhart 1995). Initially, the cells

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are positioned in the proper region by gastrulation.
Then, 5–10 h after induction by an appropriate level ofactivin A-like signal, the cells acquire a type N adhesive
property, are sorted from other cells, and stabilize in
one position. Finally, the cells differentiate from thenotochord structure and undergo secondary neuralinduction.
One intriguing finding in the present study was thatcells treated with 1 ng/mL activin A only differentiatedinto notochord and showed strong adhesive properties.
It is possible that presumptive ectoderm cells induced
by a specific level of activin A-like signal in vivo assemble to form a prenotochord thread at the site of subsequent notochord formation. It is not surprising thatcells forming the notochord, a key tissue in the developing vertebrate, should be directed by very strong
intercellular bonds.

Molecular basis of activin-induced changes in celladhesive properties
In the present study we have shown that more than
0.75 ng/mL activin A induces changes in the adhesive
properties of Xenopus animal cap cells. There are twocandidate molecules for mediation of these adhesive
properties, a heterophilic molecule like the extracellular matrix glycoprotein (ECM), or a homophilic molecule like cadherin.
The most recent studies of activin-induced changes
in cell adhesiveness have examined changes in adhesion to fibronectins (Smith et al. 1990; Howard & Smith
1993; Whittaker & DeSimone 1993; Symes et al. 1994;
Ramos & DeSimone 1996). According to those studies,blastomeres derived from induced animal pole regions
are able to spread and migrate on a fibronectin-coatedsurface in the same manner as marginal zone cells, but
unlike control animal pole blastomeres. Dispersed
animal pole cells are also able to respond to activin A
in this way (Smith et al. 1990). However, we found nodifferences in the expression of fibronectin or other
ECM molecules between activin-treated cells anduntreated cells by immunohistochemical staining (datanot shown).
The present results are generally consistent with ahomophilic alteration in adhesive properties inducedby activin A, rather than a heterophilic alteration. Cadherins, Ca2+-dependent cell–cell adhesion receptors(Nose et al. 1988; Takeichi 1991), were found to beequally expressed in activin-treated cells and untreatedcells (data not shown). Thus, there must be some otheras yet unidentified molecules mediating these changes
in adhesive properties.

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