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Transcript
Development 111, 829-844 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
829
Differential expression of two cadherins in Xenopus laevis
B. ANGRES1, A. H. J. MULLER1, J. KELLERMANN2 and P. HAUSEN 1 *
{
2
Max Planck Institut fttr Entwicklungsbiologie, Abt. fllr Zellbiologie, D-7400 Tubingen, Federal Republic of Germany
Max Planck Institut fiir Biochemie, Genzentrum, D-8033 Martinsried, Federal Republic of Germany
* Author for correspondence
Summary
Using a cadherin fraction from Xenopus tissue culture
cells as an immunogen, two monoclonal antibodies were
obtained that allowed the characterization of two
distinct cadherins in the Xenopus embryo.
The two cadherins differ in molecular weight, in their
time of appearance during development and in thenspatial pattern of expression.
One of the antigens was identified as E-cadherin. It
appears in the embryonic ectoderm during gastrulation
when epidermal differentiation commences and it
disappears from the neural plate area upon neural
induction.
The second antigen could not be allocated to any of the
known cadherin subtypes and was termed U-cadherin. It
is present in the egg and becomes deposited in newly
formed inner cell membranes during cleavage, the outer
apical membranes of the embryo remaining devoid of
the cadherin throughout development. U-cadherin is
found on membranes of all cells up to the late neurula
stages. A conspicuous polarized expression of the
antigen on the membranes of individual inner cells
suggests its participation in the segregation of cell layers
and organ anlagen.
These findings are discussed in the context of current
hypotheses on the role of cadherins in establishing the
spatial structure of the embryo.
Introduction
the aggregation of cells into distinct groups is accomplished by this differential expression of cadherins
(for review see Takeichi, 1987). Examples of this
relationship between cadherin expression and cell
sorting in early development in mouse and chicken are
the separation of mesodermal cells from the epiblast
(Thiery et al. 1984), the segregation of the neural tube
from the presumptive epidermis (Thiery et al. 1984;
Crossin et al. 1985; Hatta and Takeichi, 1986) and the
release of the neural crest cells from the neurectoderm
followed by their integration into various tissues of the
developing embryo (Aoyama et al. 1985; Hatta et al.
1987).
Most of our knowledge on the nature of cadherins
and their expression in adult and embryonic tissues is
derived from observations made on vertebrates other
than amphibians although research on amphibian
development has traditionally emphasized aspects of
cell interactions during embryogenesis. Few observations on Xenopus cadherins have been reported.
Nomura et al. (1986) demonstrated that a calciumdependent cell-cell adhesion system is operating in the
early Xenopus embryo. The trypsin sensitivity of this
system is typical for cadherins and the ability of
E-cadherin-positive F9 cells to bind to blastomeres
indicates the presence of cadherins on embryonic cells
The various ways by which cells adhere to each other
has long been a focus of attention in the discussion on
embryonic cell interactions (Townes and Holtfreter,
1955). Cell-cell adhesion is known to be mediated by
specific molecules, including the cadherins (for reviews
see Takeichi, 1988; Edelman, 1988; Kemler etal. 1989).
Cadherins represent a family of closely related transmembrane proteins that require calcium for stability
and function. Prominent members of this family are
E-cadherin (=uvomorulin), P-cadherin and N-cadherin
identified in mouse and their homologues present in
other vertebrates.
The interest in the function of the cadherins in
embryogenesis derives from their cell and tissue
specificity. Cadherin-mediated cell-cell adhesion is
accomplished by the homophilic interaction of the
extracellular domains of these transmembrane proteins. The molecular interaction between cadherin
subtypes is homotypic. When different cadherins are
present on different cells within a population, differential adhesion and cell sorting is seen to occur (for review
see Takeichi, 1990). A changing pattern of cadherin
expression has been observed in the course of
vertebrate embryogenesis. It has been proposed that
Key words: Xenopus laevis, cadherin, cell adhesion,
development, cell polarity.
830
B. Angres and others
from blastula to neurula. Levi et al. (1987) studied
cadherin expression in Xenopus embryos using an
antibody directed against L-CAM, which is thought to
be the chicken homologue of E-cadherin. A diffuse
staining within the cytoplasm of all cells up to stage 20
was observed. No preferential staining of cell membranes was seen before stage 9, in later stages some
staining of the cell borders was observed. Choi and
Gumbiner (1989) prepared monoclonal antibodies
against a putative Xenopus E-cadherin. This cadherin
was detectable in the embryo at the onset of gastrulation and was localized predominantly on cell membranes of the ectoderm.
Recently, Choi etal. (1990) and Herzberg et al. (1990)
identified antigens reacting with antibodies directed
against the intracellular domains of cadherins. The
observations indicate the presence of cadherins distinct
from E-cadherin in early stages of the embryo, but
these molecules remain poorly defined. Detrick and
coworkers (1990) reported on the appearance of
N-cadherin in the neural plate of gastrulating embryos
and suggested that this cadherin participates in the
segregation process of the neural tube.
In this communication, we report on the characterization of two different cadherins present in the early
Xenopus embryo. One of them represents the Xenopus
homologue of E-cadherin. Details of its expression
pattern complement the earlier observations of Choi
and Gumbiner (1989). It first appears during gastrulation in the ectoderm and shows a distribution pattern
that supports the hypothesis of the generation of
specific embryonic regions by differential cadherin
expression as described above.
The second cadherin (U-cadherin) is ubiquitously
expressed in all cells of the embryo from the first
cleavage up to the late neurula stages. This overall
distribution has not been described for a cadherin
before and indicates that this cadherin is present on all
cells during cell layer formation and the segregation of
the early organ anlagen. However, after organ segregation U-cadherin is localized on the border cells in a
polarized fashion with the boundary of the organ anlage
being devoid of cadherin. Cadherins may thus participate in the segregation processes in a manner more
sophisticated than previously thought.
Similarly, the polar character of the blastomeres
produced by the first cleavages is indicated by the
restriction of U-cadherin to the newly formed, i.e.
basolateral, membranes. Thus, the polarized distribution of cadherins typical for epitheha is established
from the first cleavage onwards and is maintained in the
outer cell layer of the embryo during further development.
The implications of the observations for diverse
processes of embryogenesis are discussed.
Materials and methods
Embryos
Embryos were obtained as described by Fey and Hausen
(1990) and staged according to Nieuwkoop and Faber (1967).
Cell lines and tunicamycin treatment
The A6 Xenopus kidney epithelia cell line, purchased from
the American Type Culture Collection (Maryland, USA) was
maintained in 85% RPMI 1640 (GIBCO) containing 10%
fetal calf serum (GIBCO) at 26°C in a 5 % CO2 atmosphere.
For tunicamycin treatment, a half confluent A6 monolayer
culture was incubated with lO/igmT1 tunicamycin (Sigma) in
culture medium for 22 h and used for the preparation of cell
lysates as described below.
SDS-polyacrylamide gel electrophoresis and
immunoblotting
SDS-PAGE was carried out according to Laemmli (1970).
Silver staining of gels was performed as described by
Morrissey (1981). For immunoblotting, proteins were electrophoretically transferred to nitrocellulose membranes. Blots
were blocked in 5% (or 10%) low-fat milk in PBS and
immunostained as described by Fey and Hausen (1990) with
primary antibody (ascites, diluted 1:500-1:5000) and with
secondary antibody (alkaline phosphatase or peroxidaseconjugated goat anti-rabbit IgG; Dianova). Antibody binding
was visualized with O^mgml" 1 bromo-chloro-indolylphosphate and 0.33 mgml" 1 nitroblue tetrazolium (Sigma) in 0.1 M
Na2CO3, pH10.2 or with the ECL western blot detection
system (Arnersham).
Preparation of extracts from cells and embryos for
immunoblotting
For preparation of cell lysates, one 10cm Petri dish of a
confluent grown monolayer of A6 cells or stationary grown
fibroblasts was washed three times with PBS, scraped off the
Petri dish and heated to 95 °C in 100 /il SDS-gel loading buffer
for 15min. Cell debris was sedimented by centrifugation and
the supernatant was sonicated, to fragment residual DNA.
Extracts from embryos were prepared by homogenization
at 4°C in extraction buffer (10 /A per embryo; 10 mM
Tris-HCl, pH7.4, 1.5mM CaCl2, 0.6mM MgCl2, 2% NP40)
containing the following protease inhibitors: lmM phenylmethylsulfonylfluoride, lmM N-ethylmaleimid, 0.02mM leupeptin, 0.028 mM pepstatin, lO^gml"1 aprotinin, 1 mM iodoacetamid, 1 mM benzamidin. Homogenates were centrifuged at
5000revsmin"1 and 4°C for 3min (Heraeus, minifuge). The
supernatant was extracted with an equal volume of 1,1,2
trichlorfluorethane and centrifuged in a Beckman SW65 rotor
at 100 000 £ and 4°C for 30min.
Purification of the extracellular domain of Xenopus
E-cadherin
Confluent monolayers of A6 cells were washed three times
with digestion buffer (20mM Hepes, pH7.4, 5mM KG,
150 mM NaCl) supplemented with 1.5 mM CaCl2 and 0.6 mM
MgCl2 and scraped off the Petri dishes (10 cm in diameter) in
4 ml digestion buffer containing 0.01% trypsin and either
1.5mM CaCl2 and 0.6mM MgCl2 or lmM EDTA. Cells were
incubated on a rotary shaker at 80revsmin~' and 37 °C for
45min. The trypsin digestion was stopped by addition of
150 U of Kallikrein inhibitor (Trasylol; Bayer) and cells were
pelleted by centrifugation (Heraeus, minifuge) for 5min at
2000 revs min"1 and 4°C. To remove residual cell debris the
supernatant was centrifuged in a SW 34 rotor (Sorvall) for
20min at 9000 revs min"1 and 4°C.
Supernatants of Ca2+-trypsin digestions were loaded on a
4ml lentil lectin-Sepharose column. Adsorbed material was
eluted with 200 mM o^methyl mannoside. The eluate was
concentrated and washed with PBS in a centricon 30
microconcentrator (Amicon).
Two cadherins in Xenopus laevis
Microsequencing
80 ng of the lentil-lectin-purified material was applied to a
10% SDS-PAGE according to Laemmli et al. (1970). The
separated proteins were electroblotted on a siliconized glass
fiber sheet (Glassybond, Biometra) and the 9Qx\&MT protein
band was excised and directly placed into a gas-phase
sequencer (477 A, Applied Biosystems).
Establishment of hybridoma cell lines; monoclonal
antibodies and control IgG
Protein from 32 ml of Ca2+ trypsin digest was precipitated
with trichloracetic acid and electrophoresed according to
Laemmli (1970). 90xl(fiMT protein bands were excised after
visualization with copper staining (Lee etal. 1987), destained,
equilibrated with PBS and used for the immunization of mice.
Animals with positive titers were boosted once more
intravenously with 5/ig of lentil-lectin-purified protein in
200^1 PBS. Mouse hybridoma cell lines were established as
described by Kearney et al. (1979) and Galfre' and Milstein
(1981). Inert control IgG was produced by the myeloma cell
line P3K (Horibata and Harris, 1970). Established cell lines
were used to induce ascites growth in mice. The IgG fraction
from ascites fluid was obtained by affinity chromatography on
CM Affi-Gel Blue (Biorad) as a first purification step and
subsequent ion exchange chromatography on a Mono Q
column (Pharmacia).
Immunohistology
Organs of adult frogs were frozen in liquid nitrogen; stage 47
tadpoles were fixed in 2 % TCA for 3h, embedded in Tissue
Tek (Miles Laboratories, Naperville) and frozen in liquid
nitrogen. 10pun frozen sections were cut with a Reichert Jung
microtome, mounted on gelatine-coated coverslips and fixed
in acetone at —20°C overnight. Sections were dried shortly at
—20°C and rehydrated in PBS at room temperature. Staining
was performed with a 1:500 dilution of ascites fluid.
Monoclonal antibody binding was detected with goat F(ab)2
anti-mouse IgG-FITC reagent (Dianova). Stained specimens
were mounted in Mowiol (Hoechst), examined with an
epifluorescence Axioplan Zeiss microscope.
Whole embryos of different stages and isolated dorsal
blastopore lip regions (stage 10i) were fixed in 20%
dimethylsulfoxid, 80% methanol (Dent etal. 1989) overnight
at -20°C. After several washes in PBS during 1 h at room
temperature, the vitelline membrane of whole embryos was
removed (stage 2-9) or embryos were cut into halves (stage
13-21) to improve penetration of the antibody. Specimens
were incubated with antibody (diluted in 20 % rabbit serum in
PBS) overnight at 4°C on a rocking platform followed by
washes in PBS during the day at room temperature by
changing the buffer several times. The incubation with mAb
6D5 or mAb 10H3 or inert P3 IgG (all ascites 1:500) was
followed by the incubation with FITC-conjugated goat antimouse F(ab)2 as the secondary antibody (Dianova) and a
FITC-conjugated rabbit anti-goat F(ab)2 antibody specific for
the F(ab)2 fragment (Dianova) as the tertiary antibody.
Embryos were incubated in 2 % paraformaldehyde in PBS for
l h at room temperature and washed twice with PBS for
lOmin each. Specimens were embedded in 2% agar and
dehydrated in DMP (3,2 dimethoxypropane, Muller and
Jacks, 1975) twice for 30min each. They were embedded in
glycolmethacrylate (RWL, Histotechnologie, Bruckmiihl/
Vagen, FRG) as described by the supplier. 4 fan thick sections
were cut on a Reichert Jung microtome and mounted in
Mowiol (Hoechst, FRG).
831
Aggregation assays
A6 cells were grown in 85 % Leibovitz medium containing
10 % fetal calf serum at 26 °C in microtiter plates coated with
gelatine until they reached confluency. Cells were washed
once with 85 % Leibovitz medium to remove loose cells and
incubated in 85 % PBS. After they lost intercellular contacts
but still remained attached to the bottom of the plate, cells
were incubated in 85 % Leibovitz medium at room temperature supplemented either with purified IgG of mAb 6D5 or
mAb 10H3 or with P3 IgG at a concentration of 10/igml"1
each. The reaggregation was observed with an inverted
microscope.
To test the aggregation of early cleavage blastomeres
fertilized eggs were demembranated and maintained in Ca2+and Mg^-free Modified Barth's Solution (MBS-H; 88 mM
NaCl, lmM KC1, 2.4mMNaHCO3, 0.82mM MgSO4, 0.33mM
Ca(NO3)2, 0.41mM CaCl2, 10mM Hepes (+NaOH), 1%
streptomycin, 1% penicillin, pH7.4) in a 24-well tissueculture plate coated with BSA ( 1 % solution, 1-2 h). When
eggs had produced eight dissociated blastomeres, Ca2+ and
Mg2"1" was added to the final concentration of 0.74 mM and
0.82 min, respectively. Purified IgG of mAb 6D5 or 10H3 or
P3 as a control were added to a final concentration of
lO^gml"1. The aggregation was followed with a stereomicroscope and photographs were taken after 2h (128-256 cells).
Inner animal cap cells from stage 9 blastulae were isolated
and incubated in Ca2+-Mg2+-free MBS-H containing lmM
EDTA for l h on Petri dishes coated with 1% agarose.
Dissociated cells were briefly washed in MBS-H and cells of
one embryo were incubated on 1 % agarose in microtiter wells
either in MBS-H supplemented with purified IgG of mAb
6D5, mAb 10H3 or P3 as a control at a concentration of
10/igml"1. A further control culture was maintained in the
Ca2?/Mg2+-free condition.
Results
Preparation and characterization of antibodies against
Xenopus cadherins
A property common to all cadherins is the release of
their extracellular domain from cells upon treatment
with trypsin (Hyafil et al. 1980). The released fragment
is stable to further trypsin action only in the presence of
calcium ions: omission of calcium from the digest leads
to the degradation of the cadherin fragment.
A prominent protein fragment of SHJXlO3^,. was
released from Xenopus A6 cells by trypsin incubation in
the presence of calcium (Fig. lb). This fragment was
not found in trypsin digests obtained in the absence of
calcium (Fig. la). After loading the calcium-trypsin
digest onto a lentil lectin column, the 90xlCrM r
fragment was absorbed (Fig. lc) and could be released
by cr-methyl mannoside as a rather homogeneous
fraction (Fig. Id).
Material from the lentil lectin column was further
purified by preparative SDS-PAGE and applied to a
gas-phase sequencer. The sequence of a segment of 21
amino acids at the N terminus was determined.
Comparing this sequence with the corresponding
sequences of several cadherins from other species
reveals a high degree of homology (Fig. 2).
These data indicate that the extracellular domain of
one cadherin predominates in the isolate, though they
832
B. Angres and others
a b e d
e
<90
—
f
g n
^90
m
t
Fig. 1. Characterization of the antigens from A6 cells recognized by mAbs 6D5 and 10H3. (a-d) Isolation of a 90xl(fi MT
protein fraction from supernatants of A6 cells after trypsin treatment. Supernatants from trypsin-digested A6 cells were
used for lentil lectin affinity chromatography as described under Materials and methods. Lane a: supernatant of A6 cells
treated with trypsin in the absence of Ca (500^1 per lane). Lane b: supernatant from A6 cells treated with trypsin in the
presence of Ca (500^1 per lane). Lane c: wash through of the lentil lectin chromatography (500^1 per lane). Lane d:
eluate from the lentil lectin column (300 ^il per lane which corresponds 10% of the eluate). The arrowhead indicates the
90X103 Mr protein fraction, (e-h) Identification of the 90X103 MT protein fraction by mAbs 6D5 and 10H3 on
immunoblots. Supernatants from A6 cells treated with trypsin in the presence of Ca2+ (lanes e and g) or in the absence of
Ca2+ (lanes f and h) were immunoblotted with mAb 6D5 (lanes e and f) or mAb 10H3 (lanes g and h). (i-m)
Determination of a protein moiety as epitopes for mAbs 6D5 and 10H3. Cell lysates of untreated A6 cells (lanes k and m)
or tunicamycin-pretreated A6 cells (10/igmP 1 ) were immunoblotted with mAb 6D5 (lanes i and k) or mAb 10H3 (lanes 1
and m). Approximately 100 ng of protein was loaded on each lane. Arrowheads indicate the two main protein bands
recognized by the two antibodies. Bars indicate relative molecular masses from top to bottom as follows: 200, 116, 97, 68,
43 (xlO3).
do not establish that we have purified one protein to
homogeneity.
Mice were immunized with SDS-PAGE-purified
^ x l t ^ M f fraction and hybridoma lines producing
antibodies were established. Two monoclonal antibodies, 6D5 and 10H3, were used for the further work.
Both antibodies recognized exclusively the
90X103 Mr material released from A6 cells by calciumtrypsin treatment on immunoblots (Fig. le,g). No
antigen was detected in the corresponding preparations
obtained in the absence of calcium (Fig. lf,h). When
whole-cell lysates were electrophoresed and immunoblotted, both antibodies bound to a 140x 103 Mr protein
band that probably represents the intact, uncleaved
cadherin molecule (Fig. lk,m). Antibody 10H3 also
reacted with a band of 155xl(rM r , which presumably
represents a precursor molecule, as well as with some
degradation products of lower relative molecular mass.
A6 cells were pretreated with tunicamycin to
determine whether the antibodies recognize oligosaccharide epitopes. In addition to the intact cadherin,
both antibodies recognize components of approximately 120 xlO 3 ^,. in the lysates from tunicamycintreated cells (Fig. li,l). These components presumably
represent N-linked oligosaccharide-deficient cadherins
that form in the presence of the antibiotic. Thus, it is
likely that the antibodies are directed against protein
epitopes on the cadherins.
We investigated whether the two antibodies, 6D5 and
10H3, interfere with cell-cell adhesion using the
following assay. Confluent monolayers of A6 cells were
incubated in calcium-free buffer until the cells were
released from intercellular contacts but still adhered to
the bottom of the culture dish (Fig. 3A). Calcium was
restored by replacing the buffer by normal culture
medium, supplemented either with mAb 10H3, mAb
6D5, or with control P3 IgG. Within 2 h, an epitheliumlike monolayer reformed in the presence of P3 IgG
(Fig. 3B). In the presence of either antibody, the cells
assumed a fibroblast-like morphology and did not form
the close cell contacts of an epithelium (Fig. 3C, D).
These results indicate the usefulness of the two
antibodies for functional studies of cell-cell adhesion.
Different cadherins exhibit characteristic tissue distribution. In adult Xenopus tissues, the two antibodies
stained the basolateral membrane domains of epithelial
cells preferably at the apical junctional complexes in
proximal and distal kidney tubules, and in lung
Two cadherins in Xenopus laevis
90kD
833
p.f.
75
79
60
Q E L
65
Q R L
65
Q E L
65
Q R L
65
Q E L
65
Fig. 2. Comparison of the 21 N-terminal amino acids from
a microsequence analysis of the 90X103 MT protein fraction
and the corresponding amino acid sequences of cadherins
in different species. Residues identical with those of the
90x10* MT protein fraction are enclosed in boxes. Numbers
on the right indicate % identity of the obtained 90X103 Mt
protein fraction with the listed cadherins. The N terminus
is marked by an arrowhead. X=not identified amino acid
residue; M, mouse; H, human; C, chicken; X, Xenopus;
cad, cadherin; p.f., protein fraction. Sequences from top to
bottom are reported by Nagafuchi et al. 1987 and Ringwald
et al. 1987; Wheelock et al. 1987; Gallin et al. 1987;
Miyatami et al. 1989; Nose et al. 1987; Hatta et al. 1988;
Shimoyama et al. 1989; Detrick et al. 1990.
epithelia (not shown). Sections of the head region of a
stage 47 tadpole showed an intense staining at the
basolateral cell membranes of the epidermis
(Fig. 4A,B). No staining was seen in the brain tissue.
This observation suggests that the antibodies do not
react with N-cadherin, which typically occurs in brain
tissue (Hatta et al. 1985).
The staining of liver sections with antibody 10H3
appears as pairs of parallel lines in the parenchyma
(Fig. 4D). These lines presumably indicate boundaries
of the bile canaliculi since the same staining pattern was
observed by using antibodies against cell junctional
molecules (Tsukita and Tsukita, 1989; Stevenson et al.
1986). Staining of these structures was not observed
with antibody 6D5 (Fig. 4E).
In heart tissue sections, antibody 6D5 staining
appeared as lines perpendicular to the length of cardiac
muscle cells (Fig. 4H, arrows) and as a repetitive
pattern along the side of muscle cells (Fig. 4H, arrows).
We presume that these patterns indicate staining of
intercalated discs and the costameres of the heart
muscle cells, structures that are believed to mediate
firm cohesion between the successive cellular units in
the myocardium (intercalated discs) and the physical
contact to the underlying myofibrils (costameres)
(Tsukita and Tsukita, 1989; Pardo et al. 1983). No such
structures were stained with mAb 10H3 (Fig. 4G).
Antibodies 10H3 and 6D5 recognize different
cadherins with different expression schedules in the
embryo
The finding that both antibodies stain basolateral
membrane domains and the junctional complexes in
Fig. 3. Antibody inhibition of the reaggregation of
dissociated A6 cells. Cells of an A6 monolayer culture
were dissociated in Ca2+-free buffer (A) and incubated for
2h in culture medium supplemented with either control
IgG P3 (B), purified mAb 10H3 (C) or mAb 6D5 (D) in a
concentration of 10/xgml"1 each. Bar, 50[im.
epithelia and specialized adhesive structures in nonepithelial tissues agrees with their specificity for
cadherins. The observation that specific structures in
heart and liver are each stained by only one of the two
antibodies provided the initial indication that the two
antibodies recognize different cadherins. This latter
notion was verified when the antibodies were applied in
studies of the development of the Xenopus embryo.
As in A6 cell lysates, antibody 10H3 recognized a
molecule of 140xl0 3 M r in the embryo extracts
(Fig. 5A, a and b). In contrast, antibody 6D5 recognized a protein of about 125 x l O 3 ^ in the embryo
extract, which can easily be distinguished from the
corresponding A6 cell antigen of 140x10? MT (Fig. 5A,
c and d). Thus, the two antibodies, 10H3 and 6D5,
recognize different cadherin subtypes in the embryo.
The time of appearance of both cadherins during
early development was determined by western blot
analysis of extracts obtained from embryos at different
stages ranging from the fertilized egg (stage 1) to the
late neurula (stage 20). The result is shown in Fig. 5B
and C. The cadherin recognized by mAb 10H3 was first
found in the embryo at gastrulation thereafter increasing in amount. In contrast, the antigen of mAb 6D5 was
already present at stage 1 and increased in amount up to
the gastrula stages (Fig. 5C). This differential timing of
the expression of the two antigens further strengthens
the notion that we have identified two different
cadherin molecules.
On the basis of its staining pattern in different tissues,
the molecular weight data and the time of its expression
during embryogenesis, we conclude that antibody 10H3
834
B. Angres and others
Fig. 4. Immunostainings of Xenopus tissues. Cryostat sections of the head region of a stage 47 tadpole (A-C), liver (D-F)
and heart (G-I) were immunostained with mAb 10H3 (A,D,G), mAb 6D5 (B,E,H) or inert control IgG (C,F,I). Arrows
and arrowheads inert control IgG (C,F,I). Arrows and arrowheads in H indicate the intercalated discs and the costameres
in heart tissue, respectively. Bars, 30//m.
recognizes the Xenopus homologue of E-cadherin,
which has been characterized by Choi and Gumbiner
(1989) (for further details see Discussion).
Antibody 6D5 recognizes a cadherin antigen that has
not yet been characterized in Xenopus and which
cannot currently be allocated to any of the known
subtypes of cadherins (see discussion). We will provisionally use the term U-cadherin for this component in
further discussion.
U-cadherin participates in cell-cell adhesion from
early cleavage onwards
The early presence of U-cadherin in the embryo
suggests that it might mediate cell-cell adhesion during
the cleavage stages. E-cadherin should not be involved
since it is not present at this time. Functional assays
support these assumptions. Demembranated fertilized
eggs were placed in calcium- and magnesium-free
buffer solution to avoid cell-cell adhesion between the
Two cadherins in Xenopus laevis
Fig. 5. Identification of two different cadherins in embryos
by mAbs 10H3 and 6D5. Protein extracts of embryos of
different stages were prepared as described in Materials
and methods. The equivalent of 5 embryos per lane was
used for electrophoresis and immunoblotting.
(A) Comparison of molecular masses of cadherins
recognized by mAbs 10H3 and 6D5 in protein extracts of
stage 20 embryos. A6 cell lysates (100/ig/lane, lanes a and
d) and the protein extract of stage 20 embryos (lanes b and
c) were immunoblotted with mAb 10H3 (lanes a and b) or
mAb 6D5 (lanes c and d). (B) and (C) Western blot
analysis of the antigens recognized by mAbs 10H3 (B) and
6D5 (C) during development. Stages from a-g: 1, 3, 8 10,
12, 14, 20. Numbers indicate relative molecular masses
xlO3.
a b e d
-200
-116
-97
-66
-43
B
a b c d e f g
-200
-116
-97
-66
-43
C a b c d e
835
f 9
-200
-116
-97
-66
-43
blastomeres during the first cleavages. After the third
cleavage division, the buffer was reconstituted with the
divalent cations in two of three samples. One of these
samples received antibody 6D5, the other antibody
10H3. Cell division proceeded unimpaired in all three
samples. After an additional five division cycles, the
blastomeres in the buffer free of divalent cations
formed a loose assembly. The cells were round and
formed only loose point contacts (Fig. 6A). In the
sample containing divalent cations plus antibody 10H3,
the blastomeres were in the process of aggregation. The
membranes of neighbouring cells formed broad contacts with cell shape adapting to these contacts
(Fig. 6B). The same pattern of aggregation was
observed when blastomeres were incubated with inert
P3 control IgG (not shown). In the sample containing
antibody 6D5, the appearance of the cell assembly
resembled that of the control deficient in divalent
cations, but cell contacts seemed to be tighter (Fig. 6C).
Thus, an effect of the antibody on cell aggregation was
clearly visible, but the cell contacts were not completely
obliterated. These results demonstrate that U-cadherin
is functionally active during early cleavage stages
whereas cell aggregation is not affected by the presence
of the antibody against E-cadherin.
The differential effect of the antibodies on cell
aggregation was even more pronounced in a functional
assay using disaggregated cells from the inner layer of
the blastocoel roof of stage 9 embryos. The assay was
performed in a way similar to that described for the
early blastomeres. 5h after reconstituting the medium
with divalent cations in samples containing antibody
10H3, these cells formed tight aggregates (Fig. 6E). In
samples free of divalent cations, no adhesion between
the cells was observed (Fig. 6D). In samples that had
received antibody 6D5, cell-cell adhesion was severely
impaired (Fig. 6F). The cells were competent to
associate but they remained spherical and did not form
compact aggregates as they did in the presence of
antibody 10H3.
The same experiment was performed with inner
vegetal cells, yielding the same results (not shown). To
test whether the residual adhesion in the presence of
antibody 6D5 was due to E-cadherin, the assay was
performed in the presence of both antibodies. The
result did not differ from that obtained by incubating
the cells with 6D5 alone (not shown).
The differential effect of the two antibodies on cell
adhesion supports the notion deduced from the western
blot analysis that they recognize different adhesion
molecules in the embryo. The residual aggregation that
836
B. Angres and others
Fig. 6.' Aggregation assays of blastomeres of the early embryo and inner cells of the animal cap of late blastulae. (A-C)
Fertilized eggs were demembranated and incubated in Ca2+-free buffer until the 8-cell stage was reached. The incubation
was continued in Ca2+-free buffer (A) or in Ca2+-containing buffer supplemented with 10/igmF 1 of purified mAb 10H3
(B) or mAb 6D5 (C). Photographs were taken 4h post-fertilization. (D-F) Inner cells of the animal half of a stage 9
blastula were isolated and dissociated in Ca2+-free buffer containing lmin EDTA. Cells were further incubated either in
Ca2+-free buffer (D) or in Ca2+-containing buffer supplemented with purified mAb 10H3 (E) or mAb 6D5 (F) in a
concentration of lO^gml" 1 each. Photographs were taken after 5h of incubation. By this time control embryos had
reached stage 13. Bars, 500/an.
occurs in the presence of mAb 6D5 remains to be
explained. It is conceivable that, apart from the
adhesion mediated by U-cadherin, further calciumdependent cell-cell adhesion systems are operating in
the early developmental stages.
The expression of E-cadherin is restricted to the
ectoderm and disappears from cells of the neural
plate.
Whole embryos at different stages were fixed and
immunostained, embedded in methacrylate and sectioned (see Materials and methods). With this method,
E-cadherin was first detected in the outer epithelial cells
at the original animal pole of stage 12 embryos (not
shown). In sagittal sections of stage 12.5 embryos (late
gastmla), the staining was more pronounced (Fig. 7B).
It was restricted to the ectodermal layer and extended
equally from the original animal region to the dorsal
side (including the neural plate) and to the ventral side
of the embryo (Fig. 7B). The signal intensity was
highest at the original animal region and decreased
gradually towards the vegetal pole, such that no
staining was detected at the dorsal and ventral
blastopore lips.
In stage 14 embryos (early neurula), the staining
increased in intensity as compared to stage 12.5 and was
evenly distributed in the presumptive epidermis
(Fig. 7E). Cells of the ectoderm forming the neural
plate were almost devoid of the antigen. In stage 20
embryos (late neurula), cells of the closed neural tube
Two cadherins in Xenopus laevis
837
np
Fig. 7. Distribution of E-cadherin in the embryo during early development. Embryos were immunostained with mAb 10H3
embedded in glycolmethacrylate and sectioned. A schematic drawing (A) indicates the cell layers seen in the sagittal section of
an embryo at stage 12.5 (B). The arrow in A indicates the original animal pole region. C represents a magnification of the
original animal pole region in B. (D) A schematic drawing indicates the cell layers seen in the transverse section of an embryo
stage 14 (E). (F) Transverse section of an embryo stage 20. np, neural plate; pe, presumptive epidermis; bl, blastocoel; ae,
archenteron, dbl, dorsal blastopore lip; vbl, ventral blastopore lip; be, bottle cells; no, notochord; pm, paraxial mesoderm; ar,
archenteron roof; nt, neural tube;. Bars represent in B and E 200/an, in C and F 50/an.
838
B. Angres and others
were completely unstained while the cells of the
epidermis strongly expressed E-cadherin (Fig. 7F).
At all stages, the outer, epithelial, layer of the
ectoderm was more intensely stained than the inner,
sensorial, layer. Membrane staining in the epithelial
layer was restricted to the basolateral membrane
domains, the apical membranes at the embryonic
surface were devoid of the antigen. Membranes of the
sensorial cells were uniformly decorated by the antibody.
In our interpretations, we have focussed only on
membrane staining although additional intracellular
staining has been observed. This intracellular staining,
which occurs preferentially in the nuclear area, is
difficult to interpret since a signal in this area was also
produced in controls in which the cadherin-specific
antibody was omitted from the staining procedure.
(Fig. 9B and 10D). However, the observation that the
intensity of the intracellular staining is greatly enhanced
in regions that also exhibit a clear membrane staining
might indicate the presence of some intracellular
antigen.
•
•
*
U-cadherin stains all cells from first cleavage to late
neurula
Although the western blot analysis' revealed the
expression of U-cadherin in the fertilized egg, no
staining of the egg membrane was observed (not
shown). This indicates a scattered distribution within
the egg cytoplasm of this protein before the first
cleavage division.
When the first cleavage division occurs, the cadherin
appears only on the newly formed membranes
(Fig. 8A, arrows). The original outer plasma membrane marked by the underlying pigment granules
(Fig. 8B) remains unstained. This absence of antigen on
the apical membrane of the outer embryonic cells is
maintained throughout development.
Internally, the antibody stains all cells during the
cleavage stages; no particular cell groups are marked by
the antibody, nor do specific membrane regions lack the
antigen. In Fig. 9, the distribution of the cadherin on
cell membranes of the dorsal blastopore lip region of a
stage 10i embryo is shown. Cell membranes are
decorated by the antibody in all three germ layers in
equal intensity. An intensified fluorescent signal at the
apico-lateral cell borders of the bottle cells at the
blastopore indicates a concentration of the antigen at
this site by the apical constriction (arrowhead).
Fig. 10 depicts the antigen distribution in the dorsal
region of the midgastrula, of the neural groove stage
and of the closed neural tube stage, in transverse
sections. Fig. 10A demonstrates the polar expression of
the antigen in the epithelial layers of the ectoderm and
the archenteron roof. Only the basolateral membrane
domains are stained, the apical membranes being
devoid of the antigen. In favourable instances, the
antigen is seen concentrated in the junctional complexes of these epithelia (arrows). Polar distribution of
the antigen is maintained in the neurectoderm when the
neural tube is invaginating (Fig. 10B). The inner cells
B
Fig. 8. Localization of U-cadherin to the newly inserted
cell membranes in the first cleavage stage. Embryos in the
first cleavage stage were immunostained with mAb 6D5,
embedded in glycolmethacrylate and sectioned.
(A) Immunostaining. (B) Same section in phase contrast.
Arrows indicate the staining of newly formed membranes
in the first cleavage (A). Bar, 50 jan.
initially display a more even distribution of U-cadherin
on their membranes.
Later, when the segregation of the anlagen proceeds,
a polar expression becomes obvious on internal cells as
well (Fig. 10B,C, arrows). In the anlagen of notochord,
somites and neural tube, the antigen is expressed on
membranes that contact their homotypic neighbours
within the organ anlage, but is absent from the
membrane domains that delimit the anlage from the
surrounding cells.
Two cadhehns in Xenopus laevis
839
Fig. 9. Distribution of U-cadherin in the dorsal blastopore lip region. Pieces of embryos stage 10.5 containing the dorsal
blastopore lip region were stained with mAb 6D5 (A) or control IgG P3 (B), embedded in glycolmethacrylate and
sectioned. Arrowhead indicates apically constricted bottle cells. Arrows point in the direction of the animal pole, en,
endoderm; me, mesoderm; ec, ectoderm. Bar, 100 ^m.
Discussion
Using a cadherin fraction from Xenopus A6 cells as
immunogen, two mouse monoclonal antibodies, 10H3
and 6D5, were prepared that recognize different types
of cadherin in Xenopus.
Antibody specificity
That the antibodies are specific for cadherins is based
on the following observations: both antibodies recognize a calcium-protected fragment that is released by
trypsin from the surface of whole cells; their antigens
display the typical basolateral distribution of cadherins
on epithelial cells; the antigens are also found on
specialized adhesive structures of non epithelial cells.
Further, both antibodies interfere with the calciumdependent cell-cell adhesion process in tissue culture.
Several findings provide evidence that each of the
two antibodies is directed against a different cadherin
molecule. The two antigens are of different molecular
weight in Xenopus embryos and the staining patterns of
the two antibodies in embryos were strikingly different.
Further, specialized adhesive structures found in adult
heart and liver were stained only by one antibody and
not by the other.
Neither of the two antibodies is directed against an
N-linked oligosaccharide epitope of the cadherins. The
difference in antibody specificity does not, therefore,
reflect a difference in the N-linked oligosaccharides of
the cadherins.
Interestingly, the two antibodies recognize antigens
of different molecular weight in the embryo but both
react only with a single antigen band on western blots of
A6 cell lysates. The latter seems to be a true crossreaction as the antigen precipitated from A6 cell lysates
with mAb 6D5 does react with mAb 10H3 on blots and
vice versa (data not shown). It seems that the A6 cell
antigen shares epitopes which are located on different
molecules in the embryo. This question requires further
investigations and may be solved when sequence data
are at hand.
Tissue distnbution of the antigens
One criterium that defines different subclasses of
cadherins is their pattern of occurrence in various
tissues. In the attempt to allocate the antigens of mAb
10H3 and mAb 6D5 to known subclasses, their pattern
of occurrence in Xenopus was compared to that of
cadherins in mouse and chicken.
As evident in Table 1, the expression pattern of
840
B. Angres and others
Fig. 10. Distribution of U-cadherin in neurula stages. Anterior halves of embryos stage 13 (A) and dorsal halves of
embryos stage 17 (B) and stage 21 (C and D) were stained with mAb 6D5 (A, B and C) or control IgG P3 (D). The
tissues were embedded in glycolmethacrylate and transverse sections were cut through the middle of the dorsal regions.
Arrows in B and C indicate cell membranes devoid of staining, np, neural plate; no, notochord; pm, paraxial mesoderm;
ar, archenteron roof; af, archenteron floor; so, somite; nt, neural tube. Bar, 30/on.
Two cadherins in Xenopus laevis
841
Table 1. Comparative distribution profile of different cadherins in adult tissues of Xenopus, mouse and chicken
Xenopus
Mouse
Chicken
Tissues
E-cad.
U-cad.
E-cad.1
N-cad.1
P-cad.2
L-CAM3
N-cad.4
Skin
Kidney
Lung
Liver
Heart
Brain
+
+
+
+
-
+
+
+
—
+
—
+
+2
+
+
-
n.r.
—
—
+
+
+
(+)
(+)
(+)*
-
+
+
+
+
—
—
+
+
n.r.=not reported; (+)=transiently expressed; *=expression in the epimyocardium; •Hatta et al. 1985; 2Nose and Takeichi, 1986;
Thiery et al. 1984; 4Hatta and Takeichi, 1986.
3
antigen 10H3 in adult tissues matches that of mouse
E-cadherin and its homologue L-CAM of chicken.
Antigen 10H3 is distinguished from N-cadherin by its
absence from brain and from P-cadherin by its presence
in adult Xenopus kidney and lung. P-cadherin has also
been reported to be present in mouse embryonic
endoderm and mesoderm (Nose and Takeichi, 1986),
whereas antigen 10H3 is confined to the ectodermal
germ layer in the Xenopus embryo. Similar to the case
for E-cadherin in mouse and chicken embryos, antigen
10H3 disappears from the neural plate of the Xenopus
embryo while N-cadherin begins to be expressed in this
location (Thiery et al. 1984; Nose and Takeichi, 1986;
Detrick et al. 1990).
Our data on antigen 10H3 with respect to molecular
weight, timing of expression in the embryo and
restriction to the embryonic ectoderm, agree with those
reported for Xenopus E-cadherin (Choi and Gumbiner,
1989). For these reasons, we classify the antigen of mAb
10H3 as E-cadherin.
The tissue distribution of the cadherin recognized by
mAb 6D5 does not allow a clear allocation to any of the
known types of cadherins (Table 1). It is distinguished
from E-cadherin by the observations reported here. Its
absence from the late tadpole brain tissue makes its
classification as an N-cadherin, unlikely. Mouse
P-cadherin is only transiently expressed in kidney and
lung, while antigen 6D5 is present in these tissues in the
adult frog. However, as nothing is known about the
expression pattern of P-cadherin in Xenopus, the
classification of this antigen remains unclear. A crossreaction of antibody 6D5 with further still unknown
Xenopus cadherins is possible. For these reasons, we
have provisionally chosen for the antigen of antibody
6D5 the term U-cadherin (ubiquitous in the early
embryo).
A polyclonal antiserum was made against a synthetic
peptide derived from a highly conserved region of the
cytoplasmic domain of cadherins (Choi et al. 1990).
U-cadherin may be related to an antigen recognized by
this antibody. This antigen was termed CLP (cadherinlike protein). It is present early in the embryo and its
relative molecular mass agrees with that of U-cadherin.
Similarly, Herzberg et al. (1990) described an antigen
that is recognized by an antibody directed against the
intracellular domain of mouse uvomorulin. This antigen
occurs early in the Xenopus embryo. In both reports,
the antigens are not sufficiently characterized to allow
for a full comparison with U-cadherin. The relationship
of these different components may become clear when
sequence data become available.
Spatial distribution of the cadherins raises questions
on embryonic patterning
E-cadherin appears in the embryonic ectoderm during
gastrulation at a point when other molecular markers
indicate the onset of epidermal differentiation. All of
these markers, including E-cadherin, exhibit the common feature that they are expressed in a polarized
fashion within the ectoderm, the epithelial layer being
more heavily loaded with the component than the
sensorial layer.
These epidermal markers seem to fall into two
classes. (1) A class of antigens that is expressed only
later than stage 12.5. These antigens are confined to the
prospective epidermal region, the prospective neural
tissue remaining devoid of the markers (antigen 2F7.C7
(Jones and Woodland, 1986); antigens XEPI-1,2 and 3
(Itoh et al. 1988); antigen Epi-I (Akers et al. 1986)). (2)
The other class of antigens includes an epidermal
cytokeratin gene that is expressed at pregastrula (stage
9) in the animal region of the embryo, including the
prospective neural plate region. When neural induction
commences in the dorsal ectoderm, expression of the
keratin gene is inhibited (Jamrich et al. 1987).
E-cadherin is a member of this second class. With the
most sensitive methods, we have been able to detect
E-cadherin as early as stage 10. In stage 12.5 the neural
plate area is stained with antibody 10H3 as well as the
prospective epidermis. Later, E-cadherin disappears
from the neural plate and the neural tube. These data
support the concept that neural induction deviates
ectoderm from its progression towards epidermis into
the neurogenic pathway at a time when epidermal
differentiation is already well under way. One effect of
neural induction is then seen as an inhibition of
epidermal differentiation. As neural development
progresses, this condition does not allow the expression
of the 'late' epidermal markers.
An additional similarity in the spatial expression of
epidermal cytokeratin and E-cadherin is their graded
distribution in the animal-vegetal direction in the early
phases. In both cases, the expression is initially highest
in the animal region and decreases as one moves
842
B. Angres and others
towards the vegetal pole. This pattern appears for
cytokeratin mRNA in the stage 10.5 embryo (Jamrich et
al. 1987) and for the E-cadherin molecule at the stage
12.5 embryo. At slightly later stages (stage 12 and 13,
respectively), all of the prospective epidermis expresses
the markers equivalently. In a transient phase, a wave
of epidermal differentiation seems to pass over the
ectoderm from the animal region towards the vegetal
pole.
In the mouse embryo, E-cadherin is already present
on the membrane of the egg (Vestweber et al. 1987) and
becomes restricted to the basolateral membrane
domains during compaction when epithelial cell polarity is induced by cadherin-mediated contacts (Shirayoshi et al. 1983; Johnson et al. 1986). In Xenopus,
U-cadherin, and not E-cadherin, is expressed in the
early stages of development. The deposition of
U-cadherin is restricted to the newly formed inner
membranes which represent the basolateral membrane
domains. A similar distribution has been proposed for
the Na+-K+-ATPase (Slack and Warner, 1973). These
observations underline the notion that cell polarity is a
conspicuous feature from the first cleavage division
onwards. The absence of U-cadherin allows us to define
the apical character of the outer membranes using this
molecular membrane marker.
This apical property is an early feature of the egg
membrane and is inherited by all membrane domains in
the embryo that derive from the egg plasma membrane,
i.e. the apical domains of the outer epithelium and of
the archenteron roof up to the late neurula stages. The
non-adhesiveness of the ectodermal apical membrane
domain has long been recognized (Holtfreter, 1943;
Roberson et al. 1980). We show here that this feature
may be explained by the lack of U-cadherin on these
membrane domains. The question of how the apical
character of the egg membrane arises during oogenesis
is currently being investigated (Miiller et al. unpublished data).
From egg to the beginning of gastrulation, the area
occupied by apical membrane domains does not
increase. During cleavage, there is no need for de novo
formation of outer apical membranes. Restriction of
membrane formation to the membranes in the embryo's
interior may thus maintain the apical-basolateral
polarity of the outer cells. The plasma membranes
formed in the embryo internally are generated by the
fusion of preformed Golgi-derived vesicles (Sanders
and Singal, 1975). We anticipate that these vesicles are
endowed with U-cadherin before fusion.
At stage 6 of development, the outer cells of the
embryo begin to form a functional epithelium with the
cell apices being joined by fully developed tight
junctions
(Miiller,
unpublished
observation).
E-cadherin-mediated contacts seem to be a prerequisite
for the formation of these junctions in tissue culture
(Gumbiner and Simons, 1986) and in early mouse
development (Fleming et al. 1989). In Xenopus, Ucadherin would serve this function in the pregastrula
embryo.
The inhibitory effect of antibody 6D5 on blastomere
aggregation shows that U-cadherin is a prominent mean
by which the early blastomeres are held together within
the embryo. Thus, a major function of U-cadherin in
early development is to maintain the integrity of the
embryo.
Gastrulation is accomplished by the movements of
sheets of cells with no gross intermingling of the cells
occurring within the sheets. The presence of
U-cadherin on cells of the gastrulating embryo may
facilitate the directed morphogenetic movement. When
a sheet of mesodermal cells moving in an explant across
the blastocoel roof is exposed to antibody 6D5, the cells
readily disperse and begin to move around in a nonoriented fashion (Winklbauer et al. unpublished data).
A participation of U-cadherin in the proper execution
of the gastrulation movements is inferred from this
observation. Mechanical forces generated by the
movements of cell sheets must be distributed within the
gastrulating embryo. The interconnection of the cells by
U-cadherin may serve this function.
The presence of U-cadherin on the motile cells poses
a problem at the same time. To a limited extent, the
cells do change position within one sheet in particular
during epiboly and convergent extension (Keller, 1986).
Moreover, whole cell collectives harbouring
U-cadherin slide past each other. It remains unclear
how these neighbouring cells, which are interconnected
by the homophilic interaction of the cadherin, retain
their mutual mobility. The cells obviously must possess
means to modulate cadherin function locally on their
surfaces.
During gastrulation individual regions of the embryo
begin to detach from their neighbours as they segregate
into organ anlagen. It has been postulated that these
segregation processes are accomplished by the spatially
restricted expression of different cadherins (for review
see Takeichi, 1988). The ubiquitous expression of
U-cadherin on all cells would preclude its participation
in such mechanisms. However, details in the spatial
localization of U-cadherin on individual cells reveal a
feature that may give an aid in the segregation
processes. On individual cells U-cadherin becomes
deposited in a polar manner, such that the membranes
that touch each other within the anlage carry the
antigen, whereas the cadherin is absent from the
membranes at the outer border of the segregating cell
group. The established cell group might lose adhesiveness this way. Coordinated with this cell polarization
ECM becomes deposited around the organ anlagen
(Fey and Hausen, 1990) and integrin becomes specifically expressed on the cell membranes facing the newly
formed matrix (Gawantka, unpublished observation).
Organ segregation seemingly requires the coordinated
interplay of several molecular systems, the details of
which need to be elucidated.
We thank Dr Rolf Kemler, Dr Rudolf Winklbauer and Dr
Jilrgen Behrens for helpful discussions, Ulrike Gossweiler
and Andrea Belkacemi for technical assistance, Cord Dohrmann for his help in preparing material for amino acid
sequencing, Dr David Stein for critically reading the
Two cadherins in Xenopus laevis
manuscript and Roswitha Gromke-Lutz, Angela Dressel and
Metta Riebesell for preparing photographs.
843
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{Accepted 12 December 1990)