/. Embryo/, exp. Morph. Vol. 51, pp. 63-72, 1979
Printed in Great Britain © Company of Biologists Limited 1979
63
Concanavalin A binding to amphibian embryo
and effect on morphogenesis
By J. C. BOUCAUT, 1 B. BERNARD, 2 M. AUBERY, 2 3
R. BOURRILLON 2 AND CH. HOUILLON 1
From the Labor atoire de Biologie Animate,
Universite P. et M. Curie, Paris, and
Centre de Recherches sur les Proteines, Faculte de Medecine, Paris
SUMMARY
The effect of Concanavalin A (Con A) on morphogenesis in Pleurodeles waltlii has been
studied. Embryos were incubated with various concentrations ofthelectin for a period of 6 days.
Three stages of development were examined, late blastula, young gastrula and late gastrula.
In the presence of the lectin at a concentration of 200, 150 or 100/*g/ml morphogenic
movements were delayed, altered andfinallyblocked. At lower concentrations, 50 or 25 /*g/ml,
there was a slight delay in gastrulation, but in some cases development was normal. These
findings indicate that Con A exerted an inhibitory effect on amphibian morphogenesis and
there is evidence that the lectin effect was concentration dependent.
The effects of Con A were specific since they were totally inhibited by a-methyl-D-mannopyranoside (005 M). The viability of the 24 h lect in-treated embryos was demonstrated by
washing experiments.
Labelled Con A binding to the embryos was investigated before and after discarding the
vitelline membrane. The results suggest a direct interaction between Con A and the cell
surface and this was confirmed by using fluorescein isothiocyanate Con A.
INTRODUCTION
Amphibian gastrulation is a complex morphogenic process in which all the
embryonic cells are involved (Vogt, 1929; Holtfreter, 1939). During this period,
cellular adhesiveness is markedly modified (Townes & Holtfreter, 1955; Curtis,
1961; Jones & Elsdale, 1963; Patricolo, 1967; Johnson, 1970) as are intercellular contact and cell morphology (Baker, 1965; Tarin, 1971; Monroy,
Baccetti & Denis-Donini, 1976; Smith, Osborn & Stanisstreet, 1976). Recent
studies on biological membranes have demonstrated the important role played
by carbohydrate-containing cell surface macromolecules in cellular function
(Moscona, 1971; Kleinschuster & Moscona, 1972; Collins, 1974; Marchase,
Vosbeck & Roth, 1976). Lect ins, which interact with carbohydrate-containing
1
Authors' address: Laboratoire de Biologie Animale, Universite P. et M. Curie, 9, Quai
Saint-Bernard, 75230 Paris Cedex 05, France.
2
Authors' address: Centre de Recherches sur les Proteines, Faculte de Medecine, 45, rue
des Saints-Peres, 75006 Paris France.
3
Chargee de Recherche INSERM.
5-2
64
J. C. BOUCAUT AND OTHERS
cell surface macromolecules (Sharon & Lis, 1972; Burger, 1973; Nicolson, 1974;
Bourrillon, 1975), may prove to be useful tools for the investigation of cell
surface modifications with respect to morphogenesis.
Concanavalin A (Con A) interacts with the methyl-D-mannopyranosyl
residues located in cell surface binding sites (Goldstein, Holleman & Smith,
1965; Sharon & Lis, 1972). These binding sites may play a role in embryo-cell
migration (Moscona, 1974; Krach, Green, Nicolson & Oppenheimer, 1974;
Moran, 1974<z). Con A interacts preferentially with the chordomesoderm migratory cells in the amphibian gastrula (O'Dell, Tencer, Monroy & Brachet, 1974),
and inhibits the development of Ambystoma maculatum gastrulae (Moran, 1974&).
Following our earlier studies (Boucaut, 1974; Aubery & Bourrillon, 1976),
the present report describes the effects of Con A on the morphogenic cellular
movements of Pleurodeles waltlii embryos, in relation with the lectin binding
to cell surface.
MATERIAL AND METHODS
1. Chemicals
[l-14C]Acetic anhydride (27-6 mCi/mmol) was obtained from the Radiochemical Centre (Amersham, England), a-methyl-D-mannopyranoside (a-MM)
was purchased from Sigma (St. Louis, Missouri).
2. Lectins.
Con A (Grade III) was purchased from Sigma. Fluorescein isothiocyanate
Concanavalin A (FITC-Con A) was obtained from I.B.F. (Clichy, France).
3. Labelling of Con A
Labelled Con A was prepared according to Miller & Great (1972), using
[14C]acetic anhydride. Lectin was purified by gel nitration on Biogel P 6, the
eluting buffer being 0-01 NaHCO 3 (pH 7-5) containing 0-15 M-NaCl. The specific
activity of the lectin was 104 dpm//*g. The labelled lectin behaved exactly like
native lectin in the erythroagglutination test (Bourrillon & Font, 1968). The
lectin solution was prepared at a concentration of 500 /^g/ml of eluting buffer.
4. Binding of Con A to eggs
(a) Binding of labelled Con A. Quantitative binding studies were performed in
centrifuge tubes by incubating four embryos with various concentrations of
labelled lectin (from 10 to 200 /^g/ml) in 1 ml of Steinberg's solution. After
incubation for various times at 4 °C or 18 °C, the embryos were decanted and
washed three times with Steinberg's solution (15 ml total). They were then
dissolved in 0-5 ml H2O2 110 vol for 24 h at 37 °C. The resulting solution was
added to 10 ml of A-C-S scintillation fluid (Aqueous Counting Scintillation,
Amersham, Searle, U.S.A.) and counted in a liquid scintillation spectrometer
(SL 300, Intertechnique).
Con A and morphogenesis
65
Specific binding of Con A was expressed in terms of the difference between
the quantity of lectin bound in the presence and in the absence of a-MM (0-05 M) ;
a specific binding was 10-15%.
(b) Binding of fluorescent Con A {FITC-Con A). Four embryos were incubated
with 100 /tg/ml of fluorescent Con A during a 24 h incubation at 18 °C in 1 ml
of Steinberg's solution. After incubation, the embryos were decanted and washed
three times with Steinberg's solution (15 ml total), and the fluorescence was
observed using a Zeiss universal microscope with an excitation filter KP 500
and a chromatic beam splitter FL 500.
The specificity of FITC-Con A was determined in the presence of a-MM
(005 M). The fluorescence was observed before and after removing the vitelline
membrane.
5. Embryos
For each set of experiments, the embryos were selected from a single batch.
Fertilized eggs were obtained from a stock of Pleurodeles waltlii kept at the
Laboratoire de Biologie Animale. They were reared at 18 °C and were staged
according to the instructions of Gallien & Durocher (1957). The jelly was removed
from the embryos with forceps, the embryos rinsed twice and allowed to develop
in 10 % Steinberg's solution, pH 7-8, until they reached the desired stage. Three
stages of development were investigated: late blastula (stage 7), young gastrula
(stage 8 a, small blastopore groove), and late gastrula (stage 12, small slitshaped blastopore).
6. Effect of Con A on morphogenesis.
When the embryos had reached the appropriate stage, they were transferred
and reared for 6 days in 10% Steinberg's solution containing 25, 50, 100, 150
or 200 /tg/ml of Con A.
The effects of each Con A concentration were followed on twenty embryos
from each of the developmental stages used (stage 7, stage 8, stage 12). The
embryos were observed and photographed daily throughout the incubation
period. These procedures and observations were carried out at 18 °C under
sterile conditions.
Control experiments were performed in order to test the specific action of
Con A and the viability of the treated embryos.
Inhibition assays were carried out with a-MM (0-05 M).
The solutions of lectin were pre-incubated for 30 min with a-MM (0-05 M).
To test the viability of the embryos after Con A treatment, they were washed
with 10% Steinberg's solution and allowed to develop in this culture medium.
For each experiment twenty embryos were placed in 10% Steinberg's solution
so as to study and stage normal development.
66
J. C. BOUCAUT AND OTHERS
RESULTS
1. Quantitative interaction between Con A and Pleurodeles waltlii embryos
[14C] Con A was used in quantitative assays at 4 °C (to prevent endocytosis)
in order to determine the amount of Con A bound to the eggs. The binding of
Con A increased regularly from 0 to 100 fig/m\ of lectin added and then reached
a plateau. The embryos rapidly bound labelled Con A to plateau levels within
lOmin at 4 °C, and all embryos were saturated with 100/*g/ml. No significant
differences in specifically bound labelled Con A were obtained at concentrations
of Con A ranging from 10 to 100 ^g/ml after a 10 min incubation in regard with
the age of the embryos. Thus, at saturation 0-18 ±0-06 fig, 0-13 ± 0 0 1 fig and
0-24 ± 0-09 ftg of labelled Con A were bound per embryo in the late blastula,
young gastrula and late gastrula stages, respectively. These results were obtained
from four separate and duplicate experiments. The specificity of lectin binding
was investigated in the presence of a-MM (0-05 M) which is an inhibitor of
Con A (Goldstein et al. 1965). The specific binding of the lectin reached 8590% of the total binding.
II. Effect of Con A on the morphogenesis of Pleurodeles waltlii embryos
(a) Con A was added at final concentrations ranging from 25 to 200 /*g/ml.
The development rate and the types of morphological variation were analysed
in relation to the lectin dose and time of incubation. Embryos from a control
medium without lectin developed normally (Fig. 1). Incubation with 25 /*g/ml
of Con A caused very few anomalies, even though the development time was
significantly delayed, since the embryos reached the tail-bud stage without
being malformed (Fig. 1). Following treatment with 50 fig/ml of lectin, development was retarded. The neural tube remained open indicating that a dose of
50/tg/ml was required to inhibit morphogenic development. However, all
treated embryos reached the tail-bud stage.
The effect of Con A on morphogenesis was significantly marked as the dose
was increased from 50 to 100 or 200 /*g/ml. The most striking result was the
inhibition of gastrulation and neurulation. In treated embryos developed from
the late blastula or young gastrula stage, exogastrulation occurred over a period
of 2 days. Subsequently the abnormalities observed were all non-closures of the
neural tube, ranging from neural folds separated by the subsisting yolk plug
to complete anencephaly. Finally, morphogenesis ceased during the fifth day
and the treated embryos died. Similarly, the same lectin levels (100-200 /*g/ml)
applied on late gastrula embryos caused a significant delay in neurulation, after
which development was arrested and necrosis followed.
(b) Administration of Con A doses ranging from 25 to 200 /*g/ml, associated
with a-MM (0-05 M) produced no morphological variations. In addition, no
indication of defective development was observed among the embryos incubated
Late blastula
12
12
12
O
O
o
Inhibition of
Con A effect
Exogastrulation and
neural defect
blockage at late
neumla
Gastrulation and
neurulation delay,
partial neural defect
Gastrulation and
neurulation delay
Control (normal)
Observations
200 pig
Con A
+
005 a-MM
200 pg
Con A
Con A
Con A
100 pig
50 Mg Con A
25 fig Con A
0 /ig Con A
20
Lethal
Control (normal)
Observations
Inhibition of
Con A effect
Exogastrulation and
neural defect,
blockage at late
neurula or tail-bud
stages
Gastrulation and
neurulation delay,
partial neural defect
Gastrulation and
neurulation delay
Young gastrula
Concanavalin Hours of incubation
A 0/g/ml)
48
96
Late gastrula
200 jig
Con A
+
0 0 5 a-MM
200/ig
Con A
Con A
\ 50 fig
100/ig
Con A
50 fig Con A
25 fig Con A
0 /ig Con A
20
20
Lethal
Lethal
20
Lethal
Concanavalin Hours of incubation
A (/ng/ml)
48
96
Inhibition
of
Con A
effect
Blockage at late
neurula
Neurulation delay,
neural defect
Short neurulation
delay, partial
neural defect,
tail-bud stages
Short neurulation
delay, tail-bud
stages
Control
(normal)
Observations
Fig. 1. Diagram of morphogenic alterations obtained with different concentrations of Con A. Three stages of development were studied:
late blastula, young gastrula and late gastrula. The embryos were reared in presence of Con A at various concentrations ranging from
25 to 200 /*g/ml during a 48 h and a 96 h incubation. For the study of the specificity of Con A effects, lectin was preincubated with
a-MM (005 M) during 30 min at 18 °C. The mixture was then added to the embryos and the embryo development was studied after 48 h and
96 h of incubation. The numbers on the diagram indicate the stage of development with reference to the Pleurodeles waltlii table.
200/ig
Con A
+
0 0 5 a-MM 12
200 Mg
Con A
150 jig
Con A
100 fig
Con A
50pgCon A
25 ng Con A
0 ng Con A
.Concanavalin Hours of incubation
A (/ig/ml)
48
96
o\
68
J. C. BOUCAUT AND OTHERS
Table 1. Localization of bound labelled Con A (100 fig/ml)
after a 24 h incubation
Binding of Con A (cpm/egg)
A
4°C
.18 °C
Late blastula
1140±188
Eggs with vitelline membrane
1217 + 125
Eggs without vitelline membrane
115 ±44 (9-5)
114±37(10)
Young gastrula
Eggs with vitelline membrane
1535 ±48
1356±178
Eggs without vitelline membrane
126 ±20 (8-2)
94 ±31 (7)
Late gastrula
1398±119
1395 + 54
Eggs with vitelline membrane
Eggs without vitelline membrane
100±7(7-2)
83 + 19(6)
The numbers in parenthesis indicate the percentage of labelled Con A remaining bound
after the vitelline membrane had been removed.
Each point gives the average value obtained from four separate experiments.
Con A was used at the saturation concentration (100/ig/ml).
in a-MM (0-05 M) solution alone. Therefore, these results show that the Con A
effect can be prevented by its carbohydrate inhibitor.
(c) Since morphogenesis was delayed or arrested following treatment with
Con A, the question arose whether the lectin effect was reversible. To test this,
further experiments were carried out with doses of Con A ranging from 100 to
200 /ig/ml. After incubation times of 24 h, 48 h and 72 h, the embryos were
washed and transferred to normal Steinberg's medium without Con A. Embryos
incubated in a solution containing 200/tg/ml of Con A supplemented with
a-MM (0-05 M) served as controls. The experiments gave similar results for the
stages of development choosen (late blastula, young or late gastrula). After an
incubation time of 24 h, organogenesis began, and tissue differentiation could
take place in the treated embryos. This showed that the effect of Con A can be
reversed after a 24 h treatment. In contrast, the Con A effect was not reversible
with Con A treatment for 48 and 72 h, since it was observed a total arrest of
development and death. These results suggest that as from 48 h of incubation
with Con A, the morphological modifications (exogastrulation) induced by
lectin are such that normal development could not continue. For the rest, the
development of the controls seemed absolutely normal.
III. Localization of Con A binding sites
At saturation (100 /^g/ml), the amount of bound labelled Con A was evaluated
at 18 °C after a 24 h incubation under the conditions described above. At 18 °C
the binding of labelled Con A to eggs with a vitelline membrane was similar to
those obtained at 4 °C (Table 1).
Con A and morphogenesis
Fig. 2. Binding of Con A. Young gastrula embryos were incubated in 100/*g/ml
FITC-Con A for 24 h (18 °C) and then carefully washed three times with Steinberg's
solution to remove unbound lectin. The treated embryos were observed in toto with
a fluorescent Zeiss microscope, (a) The phase contrast microscopic examination of
presumptive ectodernal cells from embryo incubated for 24 h in 100/ig/ml FITCCon A. The vitelline membrane is removed. Note the cellular periphery and the
intracellular pigments (x 400). (b) The same view as (a) under fluorescent conditions.
Note that FITC-Con A was bound to the cell surface (x 400). (c) The phase contrast
microscopic examination of presumptive ectodermal cells from embryo incubated for
24 h in 100 /Jg/ml FITC-Con A + a-MM (005 M). The vitelline membrane is removed.
Note the cellular periphery and the cell pigments (x 400). (d) The same view as (c)
under fluorescent conditions. Note the absence of fluorescence when a-MM is added.
The background of bound FITC-Con A was very low. (x400). (<?) The binding of
FLTC-Con A to the vitelline membrane (VM). The young gastrula embryo was treated
with FITC-Con A (100/tg/ml) for 24 h. Note the marked fluorescence on the
vitelline membrane (x200). (/) The same view as (e), excepted that Con A was
previously incubated with a-MM (005 M). Note the intense reduction of Con A
binding (x 200).
69
70
J. C. BOUCAUT AND OTHERS
In the presence of Con A at a final concentration of 100 /tg/ml, late blastula,
young gastrula, and late gastrula embryos bound 0-24 ± 0-03 jug, 0-31 ± 0-01 /tg
and 0-28 ± 0-01 jag, respectively. These results were obtained from four separate
and duplicate experiments.
After a 24 h incubation, the vitelline membrane was removed with forceps
and the amount of labelled Con A bound to the eggs (s.s.) was determined and
compared with the total lectin bound to the eggs with a vitelline membrane.
The results are given in Table 1. Of the total labelled Con A 7-9 % remained
bound to the eggs after the vitelline membrane had been removed. These results
suggest that Con A migrates through the vitelline membrane and that there is
a direct interaction between the lectin and embryo cell surface glycoproteins.
These results were confirmed by experiments using FITC-Con A (Fig. 2).
The fluorescent lectin bound predominantly to the vitelline membrane. However,
after the vitelline membrane had been removed, FITC-Con A remained bound
to the cell surfaces and particularly to intercellular junctions. Possibly, Con A
was evenly distributed over the blastomere surface and thus it would appear to
be more concentrated in the troughs between the cells. The interaction of FITCCon A with cell surfaces was specific since a-MM (0-05 M) prevented the binding
of Con A (Fig. 2).
DISCUSSION
The data obtained clearly demonstrate that Con A altered the development of
Pleurodeles waltlii embryos since both exogastrulation and partial neurulation
were observed. The modifications depended on the dose as well as the stage of
the treated embryo.
Our results have to be compared to those obtained by Lallier (1972), Moran
(1974(7, b) and Lee (1976) who worked on sea urchin, amphibian {Ambystoma
macuiatum) and chick embryo, respectively. Themorphogenic alterations induced
by Con A could be due to the toxicity of the lectin (Inbar, Ben-Bassat & Sachs,
1972; Nicolson, 1974). However, in our experiments this possibility was
excluded as the washing experiments clearly showed that the effect of the lectin
could be reversed, allowing tissue differentiation to take place.
These findings are in agreement with those of Lamon & Duprat (1976). The
effects of Con A could be the result of the binding of the lectin itself (Nicolson
1974). Indeed, a rapid and specific binding of labelled Con A to the eggs occurred
at 4 °C.
Slight differences in lectin binding were observed depending on the stage of
embryo development. These differences may be explained in terms of random
differences in the topography of vitelline membrane glycoproteins since the total
volume remained rather constant during the stages studied.
Although Con A was predominantly bound to the vitelline membrane, after
24 h under rearing conditions, a significant percentage (7-10 %) remained bound
Con A and morphogenesis
71
to the eggs after the membrane had been removed. Using fluorescent Con A we
were able to detect it near the 'interblastomeric' junctions, in agreement with
the results reported by O'Dell et al. (1974) on Xenopus eggs with the vitelline
membrane removed.
All these results suggest a direct interaction between Con A and cell surface
components, The well known characteristics of Con A (Goldstein et al. 1965;
Sharon & Lis, 1972) and the specificity of the binding and the effect of the lectin
as demonstrated by the use of a-MM (0-05 M) mean that cell surface glycoproteins involved in morphogenic development are Con A binding sites. The
alterations in morphogenic development induced by Con A could therefore be
correlated with the binding of lectin to cell surface glycoproteins. Con A would
inhibit the cell's relative mobility by cell to cell cross-linking and/or mask the
specific glycoproteins involved in cellular recognition and migratory activity.
We are indebted to Dr H. Denis for helpful advice and suggestions. The technical assistance
of J. Desrosiers is acknowledged.
This work was supported in part by grants from 1NSERM, ATP No 43-76-75 and CRL
No 77-1-087-3.
REFERENCES
M. & BOURRILLON, R. (1976). The action of lectins on growth of avian embryonic
cells at different stages of cell differentiation. Cell Differ. 5, 27-35.
BAKER, P. C. (1965). Fine structure and morphogenetic movements in the gastrula of the tree
frog, Hyla regilla. J. CellBiol. 24,95-116.
BOUCAUT, J. C. (1974). Etude autoradiographique de la distribution de cellules isolees
transplantees dans le blastocoele, chez l'Amphibien urodele, Pleurodeles waltlii Michah.
Ann. Embryol. Morph. 7, 7-50.
BOURRILLON, R. & FONT, J. (1968). Purification et proprietes physicochimiques d'une
phy tohemagg\utinine de Robiniapseudoacacia. lsolement d'un glycopeptide actif. Biochem.
Biophys. Acta 154, 28-39.
BOURRILLON, R. (1975). Interaction des lectines avec les membranes de surface des cellules
normales et tumorales. Biologie med. 4, 31-52.
BURGER, M. M. (1973). Surface changes in transformed cells detected by lectins. Fedn Proc.
Fedn Am. Socs. exp. Biol. 32, 91-101.
COLLINS, M. F. (1974). The role of the cell surface in embryonic morphogenesis. In Neoplasia
and Cell Differentiation (ed. B. V. Sherbet), pp. 234-278.
CURTIS, A. S. G. (1961). Timing mechanisms in the specific adhesion of cells. Expl Cell Res.
suppl. 8, 107-122.
G ALLIEN, L. & DUROCHER, M. (1957). Table chronologique du developpement chez Pleurodeles
waltlii Michah. Bull. biol. Fr. Belg. 91, 97-114.
GOLDSTEIN, I. J., HOLLEMAN, C. E. & SMITH, E. E. (1965). Protein-carbohydrate Interaction.
II. Inhibition studies on the Interaction of Concanavalin A with Polysaccharides. Biochemistry N.Y. 4, 876-883.
HOLTFRETER, J. (1939). Gewabeaffinitat, ein Mittel der embryonalen Formbildung. Arch.
exp. Zellforsch. 23, 169-209.
INBAR, M., BEN-BASSAT, M. & SACHS, L. (1972). Inhibition of ascite tumor development by
Concanavalin A. Int. J. Cancer 9, 143-149.
JOHNSON, K. E. (1970). The role of changes in cell contact behaviour in amphibian gastrulation. / . exp. Zool. 175, 391-428.
JONES, K. W. & ELSDALE, T. R. (1963). The culture of small aggregates of amphibian embryonic cells in vitro. J. Embryol. exp. Morph. 11, 135-154.
AUBERY,
72
J. C. BOUCAUT AND OTHERS
S. J. & MOSCONA, A. A. (1972). Interactions of embryonic and fetal neural
retina cells with carbohydrate binding phyto-agglutinins: cell surface changes with differentiation. Expl Cell Res. 70, 397-410.
KRACH, S. W., GREEN, A., NICOLSON, G. L. & OPPENHEIMER, S. (1974). Cell surface changes
occurring during sea urchin embryonic development monitored by quantitative agglutination with plant lectins. Expl Cell Res. 84, 191-198.
LALLIER, R. (1972). Effects of Concanavalin A on the development of sea urchin eggs.
Expl Cell Res. 72, 157-163.
LAMON, J. P. & DUPRAT, A. M. (1976). Effets de la Concanavaline A sur la morphologie el
le comportement de cellules embryonnaires d'Urodeles en differentiation in vitro. Experiential!, 1568-1572.
LEE, H. (1976). Inhibition of neurulation and interkinetic nuclear migration by Concanavalin
A in explanted early chick embryo. Devi Biol. 48, 392-99.
MARCHASE, R. B., VOSBECK, K. & ROTH, S. (1976). Intercellular adhesive specificity. Biochem.
Biophys. Ada 457, 385-416.
MILLER, I. R. & GREAT, H. (1972). Protein labeling by acetylation. Biopolymer. 11, 2533-2536.
MONROY, A., BACCETTI, B. & DENIS-DONINI, S. (1976). Morphological changes of the surface
of the egg of Xenopus laevis in the courseof development. III. Scanning electron microscopy
of gastrulation. Devi Biol. 49, 250-259.
MORAN, D. (1974o). The action of Concanavalin A on migrating and differentiating neural
crest cells. Expl Cell Res. 86, 365-73.
MORAN, D. (1974/?). The inhibitory effects of Concanavalin A on the development of the
amphibian embryo. /. exp. Zool. 188, 361-365.
MOSCONA, A. A. (1971). Embryonic and neoplastic cell surfaces: availability of receptors for
Concanavalin A and wheat germ agglutinin. Science, N.Y. Ill, 905-907.
MOSCONA, A. A. (1974). Surface specification of embryonic cells: lectin receptors, cell
recognition and specific cell ligands. In The Cell Surface in Development (ed. A. A. Moscona),
New York: J.Wiley.
NICOLSON, G. L. (1974). The interactions of lectins with animal cell surfaces. Int. Rev. Cytol.
39, 90-190.
O'DELL, D. S., TENCER, R., MONROY, A. & BRACHET, J. (1974). The pattern of Concanavalin
A binding sites during the early development of Xenopus laevis. Cell Differ. 3, 193-198.
PATRICOLO, E. (1967). Differentiation of aggregated embryonic cells of amphibians (Discoglossus pictus). Acta Embryol. Morph. exp. 10, 75-100.
SHARON, N. & Lis, H. (1972). Lectins : cell-agglutination and sugar-specific proteins. Science,
N.Y. Ill, 949-959.
SMITH, J. L., OSBORN, J. C. & STANISSTREET, M. (1976). Scanning electron microscopy of
lithium induced exogastrulae of Xenopus laevis. J. Embryol. exp. Morph. 36, 513-522.
TARIN, D. (1971). Scanning electron microscopical studies of the embryonic surface during
gastrulation and neurulation in Xenopus laevis. J. Anat. 109, 535-547.
TOWNES, P. L. & HOLTFRETER, J. (1955). Directed movements and selective adhesion of
embryonic amphibian cells. /. exp. Zool. 128, 53-120.
VOGT, W. (1929). Gestaltungsanalyse am Amphibiankeim mit ortlicher Vitalfarbung. II.
Gastrulation und Mesodermbildung bei Urodelen und Anuren. Wilhelm Roux Arch.
Entw Mech. Org. 120, 384-706.
KLEINSCHUSTER,
{Received 3 My 1978, revised 24 October 1978)