/. Embryol. exp. Morph. 86, 39-51 (1985)
Printed in Great Britain. © Company of Biologists Limited 1985
39
Target cell surface glycoconjugates and neural
induction in an amphibian
LYDIE GUALANDRIS, PIERRE ROUGE AND ANNE-MARIE DUPRAT*
Laboratoire de Biologie ginerale and Laboratoire de Biologie Cellulaire, Faculty
Sciences Pharmaceutiqu.es, Universite Paul Sabatier, 118 Route de Narbonne,
31062 Toulouse Cedex, France
SUMMARY
The possible involvement of target membrane specific receptor(s) in the transmission of the
neural signal leading to activation of the intracellular machinery involved in the process of neural
determination, has been examined using lectin probes (Con A, succinylated-ConA, LcA, PsA
and SB A). Not only Con A binding sites but many different glycoconjugated molecules (a-Dgalactose, N-acetyl-D-galactosamine, a-D-fucose, N-acetyl-D-glucosamine, etc.) would have to be
involved, if neural receptor(s) are invoked to explain initiation of neural induction. We show here
that the close involvement of such receptor molecules in neural induction is so far hypothetical
and remains to be demonstrated. Moreover we are inclined to the view of Barth and others who
suggested that ionicfluxesand physicochemical and electrophysiological properties of the target
membrane could play a crucial role in neural induction.
INTRODUCTION
The molecular mechanism of the neuralization of ectodermal cells during gastrulation is an important and yet unsolved problem of neuroembryology.
It is now recognized that a neuralizing factor (or stimulus) exerts its first effect
only at the cell surface of target cells (Tiedemann & Born, 1978; Yamamoto &
Ozawa, 1981). Several authors have shown recently the important role played by
the target cell membrane in the onset of the neural induction process (Grunz &
Staubach, 1979; Takata etal. 1981; Takato, Yamamoto, Ishii & Takahashi, 1984;
Duprat, Gualandris & Rouge, 1982; Gualandris, Rouge & Duprat, 1983). Recently,
using in vitro association of blastoporal lip with presumptive ectoderm covered
(inner side) or not (outer side) by extracellular material, we have shown that the
extracellular matrix (covering the neural target tissue) is not necessary for the
transmission of the neuralizing signal and seems therefore not directly involved in
the process of neural induction (Duprat & Gualandris, 1984).
The molecular components of the competent presumptive ectoderm surface
(neural target tissue) have a specific pattern especially in their glycoconjugated
components as visualized by binding to labelled lectin probes (Nosek, 1978;
*For reprints.
Keywords: Neural induction, cell surface glycoconjugates, lectins, amphibian embryo, cell
determination.
40
L. GUALANDRIS, P. ROUGE AND A.-M. DUPRAT
Barbieri, Sanchez & Delpino, 1980; Gualandris etal. 1983). The process of neural
induction is impaired by molecular reorganization after Soybean lectin treatment
of the target plasmalemma prior its in vitro association with the blastoporal lip
(Duprat et al. 1982). This impairment is reversed after reconstitution of the normal
molecular organization of the membrane, due to normal turnover of glycoconjugates (Gualandris etal. 1983). Therefore, glycoconjugates and /or the structural
organization of the plasma membrane of target cells play a role in the onset of the
molecular events in the neural inductive machinery which ultimately lead to neural
determination.
The aim of the present work was to discuss, in the light of new findings using lectin
probes (PsA, LcA, Con A and succinylated - Con A) which bind to the same
oligosaccharide residues (a-D-mannose, a-D-glucose residues), the hypothetical
existence of a specific neural receptor on the competent target plasma membrane
and its relationship to the molecules which bind lectins (Takata et al. 1984).
MATERIAL AND METHODS
Experimental procedure
Presumptive ectoderm was isolated from early gastrulae (stage 8) of Pleurodeles waltl staged
according to Gallien & Durocher (1957). Experiments were carried out in Holtfreter solution,
pH8-0, Tris 5mM, containing penicillin (lOOi.u.mF1) and streptomycin (lOOjugml"1).
Gastrulae were manually dejellied and the vitelline membrane removed. The microsurgically
excised ectoderm was treated with different lectin solutions (50 jug ml" 1 or 300 [ig ml"1) for 30 min
or 3 h. The explants were then washed several times in Holtfreter solution and dissociated with
Barth dissociation medium (88 mM-NaCl, 1 mM-KCL, 2-4 mM-NaHCO3,2 mM-Na2 HPO4,0-1 miuKH2 PO 4 , 0-5 mM EDTA, pH8-5). The isolated cells were cultured on dried collagen substrate
in Falcon or Nunc dishes with Barth balanced salt solution (Barth & Barth, 1959) for up to 10 days
at 20 °C. For control experiments, the ectoderm was combined with the blastoporal lip according
to the now classical Holtfreter 'sandwich-method' (Gualandris & Duprat, 1981).
The test to score neural induction was the differentiation of neurones which expressed neurofilament polypeptide markers detected by immunocytochemistry (Duprat & Gualandris, 1984).
Lectins
The lectins used (Table 1) were:
Con A - Concanavalin A (Canavalia ensiformis agglutinin)
S-Con A - succinylated Con A.
PsA - Pisum sativum agglutinin.
LcA - Lens culinaris agglutinin.
These lectins (except S-Con A supplied by IBF-France) were isolated, purified and labelled
with FITC or TRITC as previously described (Duprat et al. 1982; Gualandris et al. 1983) PsA,
LcA and Con A are known to involve the capping of membrane glycoconjugates whereas S-Con
A did not involve such a reorganization (Gunther et al. 1973).
Tests of lectin specificity
(1) Specificity of lectins for sugars
The specificity of the purified lectins was tested by an haemagglutination inhibition assay
(Table 2).
The haemagglutinating activity of native and succinylated lectins was determined by two-fold
serial dilution in 0-1 M-phosphate-buffered saline (pH7-2) on standard microtitration plates. To
Cell surface glycoconjugates and neural induction
41
Table 1. Characteristics oflectins.
number of subunits
number of sugar binding
sites per molecule
relative molecular mass
carbohydrate percentage
metal requirement
sugar specificity
Con A
S. Con A
PSA
LCA
4y4
2y2
4^202
2^2/32
4
120000
0
2
55000
0
2
49000
0-3 Glc
2
48000
2 Glc, Glc-NAc
+
+
+
+
Sugars of Makela's group III (cf. Table 2)
Glc, galactose; -NAc, N-acetyl-D-galactosamine.
each lectin solution (50 (A) were added 200/i of a 1% solution of thrice-washed rabbit erythrocytes in PBS. Agglutination was estimated macroscopically 12h later.
Inhibition of haemagglutination by sugars was tested by two-fold serial dilution in 50/ul. To
each sugar dilution 50 fA of PBS containing 50 jug ml" 1 of native or succinylated lectin were added.
After a 1 h incubation, 200 jul of a 1% solution of thrice washed rabbit erythrocytes in PBS were
added and agglutination was estimated macroscopically 12 h later.
The data of the inhibition test with simple sugars clearly indicate that Con A, S-Con A, PsA
and LcA constitute one group of lectins which bind specifically a-D-mannosyl and a-D-glucosyl
residues, a-methyl-D-mannoside being their best inhibitor.
(2) Absorption of lectins
The specificity of lectin absorption was tested by competitive inhibition. Lectins were preincubated with their hapten inhibitor (a-D-mannose) in order to prevent their haemagglutinin
activity. For a preincubation, 50/zg or 300 pig of lectin was used in lml of a 0-1M solution of
Table 2. Minimum concentration (mM) of sugar giving complete inhibition of
haemagglutination
Sugar
PsA
LcA
Con-A
S-Con A
D-mannose
D-glucose
D-fructose
D-glucosamine
a-methyl-D-glucoside
/3-methyl-D-glucoside
a-methyl-D-mannoside
N-acetyl-D-glucosamine
sucrose
6.25
25
50
100
12-5
25
50
100
200
50
—
12-5
50
50
6-25
25
25
100
3-12
—
0-78
25
25
1-56
3-12
25
50
0-78
100
0-39
6-25
6-25
3-12
25
25
The following sugars were not inhibitory at final concentration of 200 mM: D-arabinose, Lfucose, L-rhamnose and D-ribose, D-galactose, D-galactosamine, a-methyl-D-galactoside, j8
-methyl-D-galactoside, N-acetyl-D-galactosamine. The lectin concentration used (SOjugml"1) is
4- to 8-fold higher than that producing complete haemagglutination of rabbit erythrocytes at the
last 2-fold dilution.
42
L. GUALANDRIS, P. ROUGE AND A.-M. DUPRAT
inhibitory carbohydrate for 15 and 30 min at room temperature. This sugar concentration reduced
the haemagglutinin activity to zero and was sufficient to obtain maximal saturation of lectinbinding sites.
None of the biological effects of lectins were observed when the hapten inhibitor was copresent in the solution.
(3) Saturation of binding sites (SOjUgmr1 final concentration)
The saturation of lectin-binding sites on neural target cells was tested by use of unlabelled and
then fluorescent (FITC or TRITC) lectins.
Explants werefirstincubated in a solution of unlabelled lectin (Con A for example) 50 //g ml" 1
for 30 min, thoroughly washed and incubated in a solution of thefluorescentlectin (Con A-FITC
or TRITC) SOjugmF1 for 30 min. After several washings the control of the fluorescence of
explants was observed with a Leitz Dialux microscope equipped with HBO 50, filters I2 (BP
450-490; LP 515) and M2 (BP 546/14; LP 580). The saturation of binding sites for S-Con A, PsA
and LcA was tested in a similar way.
Nofluorescencewas detected on the explants. All the lectin binding sites were saturated by the
first incubation. Con A, S-Con A, PsA and LcA (50 jUgml for 30 min) were therfore in saturating concentration.
(4) Homology of binding sites
The homology of the binding sites for these four lectins studied was checked in the same way,
using first unlabelled and then labelled lectins (SOOjugmF1 for 15 or 30min).
Explants were first incubated with one lectin (Con A for example), washed, then incubated
with another lectin (PsA for example) labelled with FITC or TRITC, washed and observed in
epillumination.
All the following combinations were carried out:
1st treatment
1
washings
2nd treatment
(300 pig ml"1)
washings
Fluorescence
observation
epillumination
Fluorescent PsA
LcA
S.ConA
Observation
Con A
Fluorescent PsA
LcA
Con A
Observation
S-Con A
Fluorescent Con A
LcA
S.ConA
•*- Observation
PsA
Fluorescent Con A
S.ConA
PsA
- • Observation
LcA
In all cases, these double-labelling experiments, showed no fluorescence on the treated explants, thus indicating that PsA, LcA, S-Con A, Con A bind to the same plasmalemma-binding
sites.
(5) Tests of cell viability
In order to validate our results, the viability of cells following lectin treatments (50 and
SOOjiigml"1) was carefully checked using exclusion test with trypan-blue dye, ultrastructural
cytology, cell behaviour and differentiation over a 10-day period in vitro.
Cell surface glycoconjugates and neural induction
43
Fig. 1. Electron microscope micrograph. No nuclear or cytoplasmic abnormalities were
detected after a treatment of 24h with lectin. Bar = 0-5./xm. (N, nucleus; G, Golgi
apparatus; me, melanin granule; m, mitochondria; nm, nuclear membrane; np, nuclear
pores).
The exclusion test with trypan blue indicated a similar % of dead cells between treated and control
batches(<5%)exceptfora300jugml~1 Con A treatment (*£1O%). No cytoplasmic or nuclear abnormality was detected at an ultrastructural level after 30 min, 4 h and 24 h of treatment (Fig. 1).
Moreover in culture, the treated cells spread and differentiated normally as did the control cells.
RESULTS
Table 3 shows the percentage of neural induction in control series.
(a) Isolated gastrula ectodermal cells of P. waltl (stage 8) always differentiated into
typical epidermis (Fig. 2). No autoneuralization was observed in this species.
(b) (c) After 3 h and 4 h of association between presumptive ectoderm and blastoporal lip, neural induction occurs in approximately 80% of the cases after 3 h and
in 90% of the cases after 4 h (Fig. 3).
44
L. GUALANDRIS, P. ROUGE AND A.-M. DUPRAT
Fig. 2. Phase contrast micrograph. Culture of isolated cells from non-induced ectoderm
(cultured for 5 days): strong reaggregation and typical epidermal sheet ©. Bar =
100 jum.
Fig. 3. Phase contrast micrograph. Isolated cells from induced ectoderm cultured for
12 days: neural differentiation is observed (—•), presence of melanocytes (^).
Bar =100/an.
Cell surface glycoconjugates and neural induction
45
Table 3. Percentage of neural induction in the control association: ectoderm/blastoporal lip
Induction
Induction
frequence
126
0
0%
66
55
83%
106
97
92%
No. of cultures
(a) Culture of isolated ectodermal
cells without previous contact with
blastoporal lip (inducer).
(b) Culture of ectodermal cells
previously associated for 3 h with the
blastoporal lip.
(c) Culture of ectodermal cells
previously associated for 4 h with the
blastoporal lip.
Neural induction is checked in culture by the differentiation of neurones, immunocytochemically identified by neurofilament polypeptide markers.
(1) Effects oflectins on isolated presumptive ectoderm (stage 8)
(A) Lectin-treatments for 3 h, 50 fig ml ~*
These experiments were performed in saturating conditions for lectin-binding
sites on target cells. Table 4 shows the absence of an inducing effect by lectins
themselves. The treated cells behaved and differentiated in the same way as control
cells (Table 3, batch a) into epidermal cells (Fig. 4). Under saturating concentration
of SOjUgml"1 for 3 h, none of the studied lectins had neural inducing properties.
(B) Lectin treatments for 3 h, 300 fig ml'1
Table 5 shows the percentage of neural induction occurring after treatment of
presumptive ectoderm with lectins (at a high concentration),
(a) Con A at high concentration (300 //gmr 1 for 3h) induced neural structures in
80% of the cases (differentiation of neurones, (Fig. 5), melanocytes, etc. could be
easily observed). These results were in agreement with those obtained by Takata
etal. (1981, 1984).
Table 4. Comparison of the inducing effect of lectins, 50 fig ml'1.3h
neural target tissue (presumptive ectoderm).
on isolated
Lectins
Sh)
Con A
S-Con A
PSA
LCA
No. of cultures
Induction
Induction frequency
35
46
57
30
0
0
0
0
0%
0%
0%
0%
The absence of neural induction is observed in all series.
46
L. GUALANDRIS, P. ROUGE AND A.-M. DUPRAT
Table 5. Comparison of the inducing effect of lectins, 300 pig ml*1,3h
neural target tissue (presumptive ectoderm).
on isolated
Lectins
1
(a)
(b)
(c)
(d)
.3h)
Con A
S-ConA
PSA
LCA
No. of cultures
Induction
Induction frequency
51
60
31
29
41
0
0
0
80%
0%
0%
0%
Only Con A treatment provokes neural induction.
(b) No ectoderms treated with S-Con A were induced, only epidermal differen-
tiation was observed. Thus S-Con A did not exhibit a neuralizing effect.
(c) (d) No neutralization could be detected when gastrula ectoderm was treated
with PsA or LcA. In both these series, all cells differentiated into epidermal
cells in the same way as after S-Con A treatment and for the non-induced control
series.
Among the four studied lectins which bind to oligosaccharides with mannose and
glucose residues, only Con A had a neuralizing effect.
(2) Lectin effects on neural induction obtained by association of presumptive
ectoderm and blastoporal lip
We have previously observed (Duprat et al. 1982) that the treatment of presumptive ectoderm by PsA or SBA (soybean agglutinin) prior to its association with
blastoporal lip, led to a large reduction in the percentage of neural induction (10%
of induction after SBA treatment, 20% for PsA treatment; control experiment
without treatment: 90%). Table 6 shows the percentage of induction obtained
when the ectoderm was preincubated for 30 min with Con A or S-Con A or LcA
(50/igml" 1 ) prior to association for 4 h with blastoporal lip.
In the same way as for PsA, LcA inhibited the inductive machinery when the
treated ectoderm was associated with the natural inducer. The induction frequency
fell from 90% in the control batch to 19% in the treated batch. Con A and S-Con
A did not have such an inhibitory effect.
Fig. 4. Electron micrograph. Cultured cells from target neural tissue (presumptive
ectoderm) treated with ConA SOjUgml"1 for 3h: the behaviour and differentiation of
treated cells were identical to non-induced control cells (Fig. 2), only epidermal differentiation is observed. Bar = 1-35 jum. (c, cilia; mv, microvilli; ecm, extracellular
material; desmosomes (—-^-), m, melanin granule; L, lipid droplet).
Fig. 5. Electron micrograph. Neural induction occurs when presumptive ectoderm is
treated with Con A SOOjUgml"1 for 3h. Bar = 0-5jum. (nf, neurofilaments; nt,
neurotubules; dv, dense cored vesicles; cv, clear vesicles).
Cell surface glycoconjugates and neural induction
#&**•$$?•
?.
&
Figs 4-5
47
48
L. GUALANDRIS, P. ROUGE AND A.-M. DUPRAT
Table 6. Effects oflectins on neural induction involved by the blastoporal lip.
Lectin pretreatment
(SO^gmr^SOmin)
prior association to the blastoporal
lip for 4 h
No. of cultures
Induction
Induction
frequency
(a) LcA
(b) Con A
(c) S-ConA
21
30
16
4
22
15
19%
72%
92%
PsA
SBA
86
79
17
7
19-7 %
8-8%
To summarize these experiments, the effects of four lectins with affinity for the
same carbohydrate residues were studied under saturating conditions:
(A) PsA and LcA were not found to have neural inducing properties (50 /ig ml" 1
and SOOjUgmr1). Moreover as previously reported they reversibly inhibited the
process of induction by the natural inducing tissue (Gualandris et al. 1983).
(B) Con A was not a neural inducer at 50 /ig ml" 1 (saturating concentration) and
moreover did not prevent neural induction by the blastoporal lip. As opposed to
PsA and LcA, when used at high concentration (300 //g ml"1) it had inducing
properties.
(C) S-Con A like PsA and LcA did not present an inducing effect (50/ig and
300jUgml~1). It did not inhibit the natural inductive process.
DISCUSSION
On amphibian neural target tissue, the use of double-labelled (FITC or TRITC)
lectin probes, as well as the experiments with hapten inhibitors, suggested that
these lectins react with identical carbohydrate residues on the cell surface. Con A,
S-Con A, PsA and LcA are therefore assumed to bind to a common structure on
the plasma membrane.
Under rigorously identical conditions (300/igml" 1 for 3h) only Con A had a
neuralizing effect on the competent presumptive ectoderm (induction in 80% of
cases). Although the occurrence of this inducing action as the result of a cytolytic
effect of high concentration of Con A cannot be totally excluded (^ 10% dead
cells), this inducing effect of Con A seems not to be such a consequence since a
similar death rate was sometimes found in control cultures without involving neural
induction.
Several comments arose from the results obtained in these experiments. If we
accept the hypothesis of neural receptor existence for the neural inducing signal;
Con A binding to a-D-mannose and glucose-containing sites, then such glycoconjugates would be good candidates for such a role (Takata et al. 1981, 1984).
Nevertheless the saturation of these sites with S-Con A, a dimeric chemical
Cell surface glycoconjugates and neural induction
49
derivative of Con A which binds to the same sugar residues, does not lead to an
inducing effect. Likewise saturation with other lectins (PsA and LcA) does not lead
to induction either. In addition, Con A has no inducing properties at lower concentrations (50 fig ml" 1 for 3 h) although the competitive inhibition method showed
that all binding sites were saturated.
Another possible explanation of these results lies perhaps in the fact that two
kinds of lectin-binding sites might exist on the membrane surface: (A) main sites
unrelated to induction, to which all the tested lectins would be strongly directed;
(B) weak-binding sites related to induction which only bind Con A at sufficient
concentration.
However, this hypothesis cannot explain why S-Con A whose sugar specificity is
closely related to that of Con A, remains ineffective.
Moreover, in the experiments in vitro on the association of target tissue
(presumptive ectoderm) with the natural inducer (blastoporal lip) we observed that
pretreatment of the target tissue with SBA, PsA or LcA gave rise to a failure of
neural induction in explants. Although these experiments cannot indicate whether
it is a problem of receiving the stimulus or giving the response, if the inductive signal
requires specific membrane receptor(s), these lectin-binding molecules could be
directly concerned; thus many different glycoconjugates would seem to be involved,
namely: a-D-galactose, N-acetyl-a-D-galactosamine, a-D-mannose, a-D-glucose,
a-D-glucosamine, etc.
The possibility of close involvement of oligosaccharides in the neural inducing
mechanism itself and the existence of such neural specific receptor (s) is still hypothetical and there is as yet no direct evidence for them.
Whatever the hypothesis one can suppose that it is not the binding itself of Con
A which initiates neural induction but that Con A possesses properties over and
above those of S-Con A, PsA, LcA, which could be responsible for this inducing
effect.
In this respect, due to the fact that only tetravalent Con A but not divalent Con
A produces neural induction, is the crosslinking of the cell-surface-binding sites due
to the multivalence of this lectin (Trowbridge, 1973, Gunther et al. 1973) involved
for this inducing activity?
Moreover, it had been shown that Con A involved ionic fluxes in treated cells.
Thus Inoue et al. (1977) reported that Con A but not divalent S-Con A caused a
marked induction of K + release from cells such as rabbit reticulocytes, Wolff &
Akerman (1982), Dufresne-Dube, Metivier, Dube & Guerrier (1983) have demonstrated that Con A elicites Ca2+ fluxes. On the other hand, in the light of experiments performed on Ranapipiens gastrulae, Barth (1965,1966), Barth and Barth
(1967,1968 and 1974) proposed the following scheme for neural induction:- 'during
gastrulation, ion (Na + , K + , Ca 2+ , Mg2+) diffusing from cells are trapped between
the two surfaces of the ectoderm and the underlying chordamesoderm. It would be
the resulting increase in concentration of ions which could initiate induction of
neural plate'. Warner and coll. (for reviews see Warner, 1984) have shown in
50
L. GUALANDRIS, P. ROUGE AND A.-M. DUPRAT
Xenopus neurulae that the intracellular concentration of cations (Na + , K + ) controls
neuronal differentiation. It is not yet clear if these changes in cation content act as
a trigger or whether they are a co-factor. A stimulating effect of the cation
ionophore A 23187 on in vitro neuroblast differentiation has also been observed in
Pleurodeles waltl (Duprat & Kan, 1981). Recently Stern (1984) has proposed an
interesting model for early morphogenesis involving an ionic mechanism.
Whatever the mechanisms of action of the numerous inducing factors known up
until now, it is therefore quite possible that the competent target tissue itself contains
the capacity and the specificity needed for neural induction. All that these neuralizing
factors so far studied would have in common is the capability to initiate the same
signal which sets in motion the machinery of neural determination.
Modification in membrane potentials and/or the initiation of ionic fluxes could
be crucial factors in this process.
This work was supported by grants from the CNRS and the M.E.N. We thank Dr. S. Jarman
for reviewing the English manuscript.
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(Accepted 14 November 1984)