/ . Embryol. exp. Morph. Vol. 70, pp. 171-187, 1982
Printed in Great Britain © Company of Biologists Limited 1982
Neural induction and the structure of the
target cell surface
By A. M. DUPRAT, 1 L. GUALANDRIS AND P. ROUGE
From the Laboratories de Biologie generate (ERA-CNRS n° 327) et de
Biologie cellulaire (Faculte des Sci. Pharm.), Universite Paul Sabatier
SUMMARY
Lectins (SBA and PSA) were used to provoke crowding and structural modifications of the
presumptive ectoderm cell surface in order to investigate the role of the membrane organization of the competent target cells in neural induction. Are specific characteristics of the
cell surface essential for this phenomenon to occur?
From amphibian gastrulae, it is possible to obtain neural induction in vitro by association
of presumptive ectoderm (target cells) with chordamesoderm (inductor tissue): 4 h of contact
is sufficient in Pleurodeles waltl for transmission of the inductive signal.
Very quickly, the treatment of the normal ectoderm by lectins (SBA-FITC or PSA-FITC)
provoked surface modifications.
Lectin-treatment (50 fig ml"1, 30 min) of presumptive ectoderm did not result in any
neural induction.
Lectin-treatment (50 fig ml"1, 30 min) of presumptive ectoderm previous to its association
with the natural inductor for 4 h, disturbed the phenomenon: no induction.
Similar treatment followed by association with the inductor for 24 h: induction.
Treatment of SBA or PSA with their respective hapten inhibitors prior to addition to
ectodermal cells completely blocked the suppressive effects on induction.
The structural integrity of the membrane of competent target cells is necessary for neural
induction to occur. The cell membrane could thus play, directly or indirectly, an active role in
the specificity of this process.
INTRODUCTION
Neural induction is an epigenetic process which depends on the capacity of
the blastoporal lip to induce the ectoderm coming in contact with it during
gastrulation. The capacity to stimulate neuralization in competent gastrula
presumptive ectoderm is not restricted to normal inducing tissue. Various
tissues and apparently very different factors are inducers (cf. Saxen & Toivonen,
1962; Yamada, 1981; for review). Recently Tiedemann & Born (1978) showed
that a neuralizing factor, isolated in their laboratory, remains biologically
active on ectoderm even when covalently bound to CNBr-Sepharose. All these
data suggest an important role of the target cell membrane, which seems to
be important for the specificity of this phenomenon.
1
Author's address: Laboratoire de Biologie generate, Univ. Paul Sabatier, 118, route
de Narbonne, 31062 Toulouse Cedex, France.
172
A. M. DUPRAT, L. GUALANDRIS AND P. ROUGE
In agreement with Yamada (1981), we thought 'this area of research now
entering a new exciting phase, in which contemporary ideas and techniques
can be applied to solving the old problem'.. .An experimental approach to
the possible role of the target cell surface conformation in neural induction
consists in disorganizing the structure of the plasmalemna of competent
ectoderm and then associating the latter with natural inductor. Will neural
induction occur or not?
Under carefully controlled conditions, lectins are useful probes of the roles
of cell surface glycoconjugates in biological interactions. One of the properties
of lectins is to bind selectively to specific carbohydrate residues of the cell
surface (Cf. Nicolson, 1974; Sharon, 1977) and bring on patching and capping
reorganization of glycoconjugate elements in transformed cells (Aub, Sanford
& Cote, 1965; Burger, 1969, 1973; Nicolson, 1973; Nooman & Burger, 1973;
Bourrillon, 1975) and embryonic cells (Imbar & Sachs, 1973; Johnson &
Smith, 1976, 1977; Zalik & Cook, 1976; Nosek, 1978; Barbieri, Sanchez &
Delpino, 1980).
In order to investigate this phenomenon two complementary lectins were
used; Soybean lectin (SBA) which binds iV-acetylgalactosamine or galactose
and Pea lectin (PSA) which has a complex binding requirement that involves
AT-acetylglucosamine mannose and fucose residues.
MATERIALS AND METHODS
(1) Isolation of lectins
Soybean (Glycine max (L.) Merr., cv Hodgson) and garden pea (Pisum sativum
L., cv petit provencal nain) were used for lectin isolation.
CH-Sepharose 4B (which is formed by covalent linkage of 6-amino hexanoic
acid to CNBr-activated Sepharose 4B) and Sephadex GlOO were products of
Pharmacia, Uppsala, Sweden. l-ethyl-3-(3-dimethylaminopropyl)carbodiimideHC1 was obtained from Merck, Darmstadt, West Germany. Fluorescein isothiocyanate (FITC, isomer I) and purified bovine serum albumin were obtained
from Sigma, St Louis, U.S.A. Acrylamide, bis-acrylamide, temed and Coomassie brilliant blue R were from BioRad, Richmond, U.S.A. All other reagents
were commercial preparations of Merck pro analysis grade.
Soybean lectin (SBA) was purified by affinity chromatography on SepharoseiV-caproylgalactosamine prepared according to Allen & Neuberger (1975).
Soybean meal (10 g), thoroughly defatted with light petroleum, was extracted
overnight with phosphate-buffered saline (PBS, 200 ml) at 4 °C and centrifuged
at 15000^ for 20 min. The supernatant was applied to a column of Sepharose7V-caproylgalactosamine (5x1-6 cm), previously equilibrated at room temperature with PBS. After elution of a main peak containing almost all the
meal protein, the column was extensively washed with PBS until the absorbance
of the effluent remained constant and below 005 at 280 nm. Lectin was then
Target cell surface in neural induction
173
•
2
+
Fig. 1. Purity of PSA(1) and SBA<2) by polyacrylamide gel electrophoresis.
eluted by adding 0 1 M galactose to PBS and the lectin-containing fractions
were pooled, dialysed extensively against PBS at 4 °C, and frozen at - 30 °C
until analysis but for no more than 2 months to prevent a possible molecular
aggregation.
Garden pea lectin (PSA) was isolated by affinity chromatography on Sephadex
G 100 (Agrawal & Goldstein, 1967). Seed flour (50 g) was extracted overnight
at 4°C with 015M-NaCl, 0 05MTris-HCl buffer pH 7-6 (250 ml) and the
mixture was centrifuged at 15000g for 20 min. Ammonium sulphate was
added to the supernatant. The proteins precipitated between 30-60% salt
saturation were collected by centrifugation, dissolved in 200 ml of Tris buffer
and extensively dialysed against Tris buffer. Purification of the PSA was
achieved by filtering the protein extract through a Sephadex G100 Column
(70x2-6 cm), equilibrated with Tris buffer. The first peak was discarded and
a second peak corresponding to the PSA was eluted with 0-1 M glucose. The
174
A. M. DUPRAT, L. GUALANDRIS AND P. ROUGE
eluted lectin was dialysed against several changes of Tris buffer to remove
glucose then stored frozen at - 30 °C.
The purity of the SB A and PSA preparations (Fig. 1) was checked by
polyacrylamide gel electrophoresis according to Davis (1964), at a constant
current of 2 mA/gel. Protein fractions were fixed and stained with Coomassie
brilliant blue according to Chrambach, Reisfeld, Wyckoff & Zaccari (1967).
At basic pH PSA shows two main bands corresponding to isolectins previously described by Entlicher, Kostir & Kocourek (1970). In the same conditions SBA also shows two bands while at acid pH only a single band is
obtained. Obviously the above data are not correlated with the polypeptide
compositions of the lectins since SBA is a tetramer of almost identical subunits,
while PSA is a tetramer consisting of two types of markedly different subunits
(Lis & Sharon, 1981).
The protein content of the SBA and PSA preparations was estimated by
the microbiuret procedure of Goa (1953) using a standard of bovine serum
albumin.
(2) Labelling of lectins
Fluorescent-labelled lectins (FITC-SBA and FITC-PSA) were prepared
according to the slightly modified procedure of The & Feltkamp (1970). 1 ml
of 0 1 M phosphate buffer (pH9-5) containing 10 mg of lectin was incubated
at 25 °C and 250 /A of phosphate buffer containing 200 fi% of FITC were added
under continuous stirring. The mixture was incubated for 4 h at 25 °C and
then the FITC-labelled lectin was isolated and separated from non-bound
FITC by affinity chromatography either on Sepharose-iV-caproylglucosamine
(FITC-SBA) or on Sephadex G 100 (FITC-PSA) as previously described. In
both cases, FITC-labelled lectins were extensively dialysed against PBS or
Tris buffer to remove galactose or glucose then stored frozen at - 30 °C.
As in the case of unlabelled lectins, the purity of the FITC-labelled lectins
was checked by polyacrylamide gel electrophoresis.
(3) Lectin inhibition
SBA and PSA being inhibited by Makela group II and group III sugars,
D-galactose and D-glucose were used to inhibit SBA and PSA respectively.
These sugars were used at a concentration of 2 % (w/v) at which the haemagglutinating activity of the lectins reduced to zero.
(4) Culture methods
Gastrulae (stage 8) of naturally laid eggs from Pleurodeles waltl were
staged according to the table of development established by Gallien & Durocher
(1957). Several different lays of eggs were used.
After removal of the jelly coat and vitelline membrane, the competent
Target cell surface in neural induction
175
presumptive ectoderm and the blastoporal lip were microsurgically excised
in Holtfreter solution including penicillin (100 i.u. ml"1), streptomycin (100 fig
ml"1) and buffered with Tris 5 mM pH 8. Neural induction was produced in vitro
using the classical sandwich method (Holtfreter, 1933). Before its association
with ectoderm, blastoporal lip was maintained for 15-20 min in Holtfreter
medium to permit it to form a ball (easier to remove from the sandwich).
Afterwards, the samples were divided in two batches:
(1) Ectoderm explants were cultured at 21 °C for several days: generally
for 10 days. They were then fixed in Helly's fixative, embedded in paraplast,
sectioned (7-5 /im), stained by the Unna technique and examined histologically.
(2) Ectoderm was dissociated into a single cell suspension with Barth dissociation medium (NaCl, 88 mM; KC1, 1 mM; NaHCO 3 , 2-4 mM; Na 2 HPO 4 ,
20mM; KH 2 PO 4 , 0 1 mM; EDTA, 0-5 mM). The isolated cells were cultured
in Barth balanced salt solution (1959) including antibiotics, in Petri dishes
(Falcon or Corning) coated with collagen substrate, for 10 days or more, at
21 °C (Gualandris & Duprat, 1981). We developed this new technique to
allow earlier checking of the morphological result of neural induction. Its
advantages are: (1) The occurrence or the absence of induction can be quickly
judged, within approximately 48 h if cell spreading is considered or 4-5 days
if morphological differentiation is observed (cf. Gualandris & Duprat, 1981).
(2) It is easy to observe an eventual contamination by myoblasts or chordal
cells and then eliminate this culture (8 contaminated/197 cultures).
This technique presents several advantages complementary to those offered
by the classical sandwich method; it offers the following possibilities: (1) the
daily progress of the morphological events can be monitored; (2) labelled
precursors ([3H]choline or [3H]tyrosine), easily incorporated into the isolated
cells from the culture medium, allow determination of whether the morphologically differentiated neuroblasts are functional and synthesize neurotransmitters (acetylcholine, catecholamines); (3) in situ cytochemical analysis can
complement the biochemical study.
(5) Lectin treatments
Lectin solutions (Holtfreter medium) were prepared immediately before use.
Preliminary experiments were performed to establish the 'dose-response':
concentrations of 1, 3, 10, 25, 50, lOO/tgrnl"1 were studied. Optimal concentrations were found to be 25 or 50 /*g ml" 1 for 30 min treatment. Higher
concentrations were toxic for the explants and in vivo cytological anomalies
were observed in living dissociated cells (nucleolar and chromatin alterations,
cytoplasmic anomalies).
Presumptive ectoderm explants incubated in lectin solutions for 30 min at
room temperature, washed in Holtfreter medium, were then associated with
blastoporal lip for 4 h or 24 h and more.
Competitive inhibition. Previous to treatment, competitive inhibition (30 min)
176
A. M. DUPRAT, L. GUALANDRIS AND P. ROUGE
Presumptive
Ectoderm
w
Blastoporal lip
No treatment: control
or
(lectin
Treatment) SU^T
* lectin + sugar
Fluorescence observ.
O
Waiting in
Holtfreter medium
Association
Ectod./Bl. lip
O Removal of Bl. lip
Dissociation
of Ectod.
Culture
Dissociated Ectod.
cells
In toto explant
Fig. 2. Experimental procedure.
between SBA/a-D-galactose 0 1 M (2%) and PSA/a-D-mannose 0 1 M (2%)
were performed to check the lectin specificity.
(6) FITC-conjugated lectins
FITC-SBA or FITC-PSA were used in the same way.
The cell surface binding sites and the changes in fluorescence patterns were
observed in vivo or in whole explants immediately after the beginning of
treatment and for the following hours (up to 24 h) with Leitz-Dialux (equipped
with HB 050, Orthomat).
All these experiments are summarized in Fig. 2.
RESULTS
(I) Contact-period necessary for transmission of inductive signal
in Pleurodeles waltl
The minimum contact duration for neural induction should be accurately
defined. Papers on these studies give very varying results (cf. Denis, 1956).
Table 1 shows the results we obtained.
Target cell surface in neural induction
177
Fig. 3. Histological technique: (a) Non-induced presumptive ectoderm cultured
10 days: only epidermal cells differentiate; (b) Induced ectoderm cultured 8 days;
neural differentiation is observed: neural tube (N.T.), secondary induction (S.I.).
Cell culture: (c) Isolated cells from non-induced ectoderm cultured for 3 days: strong
reaggregation and typical epidermal sheet, (d) Isolated cells from induced ectoderm,
cultured 5 days: here again neural differentiation is observed (N), note the presence
of melanophores (M).
178
A. M. DUPRAT, L. GUALANDRIS AND P. ROUGE
Table 1. Contact period necessary for transmission of
inductive signal in Pleurodeles waltl
Ectod.
dissociated
cell culture
Contact period
Ectod./blast. lip
0 min
10 min
15 min
20 min
30 min
lh
2 h 15 min
3h
4h
6h
24 h and
more (control)
In i oto
ectoderm
-\
No. of
No. of
explants Induc- explants Induction
tion
67
9
16
6
31
12
31
64
44
24
59
0
0
0
0
0
1
11
52
40
24
59
24
4
9
2
8
10
14
2
6
3
12
Induction frequency
(%)
(0/91)
(0/13)
(0/25)
(0/8)
(0/39)
(1/22)
(15/45)
(55/66)
(44/50)
(27/27)
(71/71)
0
0
0
0
0
0
0
0
0
0
0
4
0
4
3
12
0045
33-3
78-8
88
100
100
In both methods of culture used, we noted that: (1) as before (cf. Gualandris
& Duprat, 1981), results were similar for the two techniques (Fig. 3a, b, c, d);
(2) in Pleurodeles waltl a 4 h blastoporal lip/ectoderm contact seems to be
satisfactory in order to obtain approximately 90 % neural induction.
Table 2. Soybean lectin (SBA) effects on neural induction
Contact period with
blast, lip after 30 min
ectod. lectin treatment
(a)
(b)
(c)
Oh
4h
24 h
No. of
explants Induction
20
79
16
0
7
16
Induction frequency
100
(0/20)
(7/79)
06/16)
(II) Lectin effects
(A) Changes in surface structure
Presumptive ectoderm explants were treated in vivo with labelled fluorescent
lectins (SBA or PSA) in order to follow the binding to the surface sites and
to observe their behaviour.
Non-treated explants did not show any autonomous fluorescence.
Treated explants first showed a fluorescent line around the cells (Fig. 4a).
Very quickly (2 to 3 min) fluorescent clusters and caps appeared on each cell
(Fig. 4 b). These patterns were to be observed for several hours. This process
Target cell surface in neural induction
179
Fig. 4. Presumptive ectoderm treated with fluorescent SBA (50 fig ml"1, 20 °C,
5 min). (a) First, fluorescent line appears only around the cells; (b) Patches and
caps appeared after, indicating surface modifications.
180
A. M. DUPRAT, L. GUALANDRIS AND P. ROUGE
Fig. 5. Ectoderm treated with SBA (50/fgml-1) associated with inductor tissue
4 h: neural induction is inhibited, only epidermal differentiation is observed in cell
culture.
Fig. 6. Ectoderm treated with SBA (50 /tg ml-1) associated with inductor tissue
24 h: neural induction occurs normally (cell culture).
Target cell surface in neural induction
181
Table 3. Pisum sativum lectin (PSA) effects on neural induction
Contact period with
blast, lip after 30 min
ectod. lectin treatment
(a)
(b)
(c)
Oh
4h
24 h
No. of
explants
Induction
Induction frequency
57
86
16
0
17
16
0
19-7
100
(0/57)
(17/86)
(16/16)
seemed irreversible when explants (5 or 30 min treatment) were then maintained
in medium without lectin. Lectins fixed then remained fixed on ectoderm for
several hours. The structural integrity of the target cell surface was very
quickly and strongly modified by lectins.
(B) Effects on neural induction
Table 2 summarizes the experiments performed.
(i) Soybean lectin
(a) In ectoderm explants treated with SBA (50 fig ml"1, 30 min) and cultured
for 2 to 3 weeks lectin had no neuralizing effect. The ectoderm presented typical
epidermal differentiation (Table 2 a).
(b) Ectoderm explants treated with SBA (50 /tg ml"1, 30 min) were associated
with blastoporal lip for 4 h. The blastoporal lip was then removed and the
ectoderm cultured for several days. Neural induction was strongly inhibited'. 51
inhibited explants: 55 explants in all (Fig. 5; Table 2b). Control experiments
performed in the same conditions, without lectin, provided normal neural
differentiation.
(c) Ectoderm treated with SBA (50 jug ml"1, 30 min) was associated with
blastoporal lip for 24 h or more. In these cases, neural induction took place
and normal differentiation was observed (Fig. 6; Table 2c).
(d) Blastoporal lip treated in the same way, by lectin, did not lose its
inductive capacity.
It is important to underline that the dose of SBA used (50 /*g ml"1, 30 min)
was not toxic: (1) treated cells lived as long as controls; (2) no nuclear or
cytoplasmic anomalies were observed in these cells, no cytolysis and no
abnormal behaviour (observations on living cells and ultrastructural studies);
(3) 24 h after treatment, cells were able to be induced and develop normally
(i.e. identically to the control).
(ii) Pisum sativum lectin
The observations were similar to SBA effects (Table 3).
(a) The control experiment showed the absence of neuralizing effect on
competent presumptive ectoderm (Table 3 a).
182
A. M. DUPRAT, L. GUALANDRIS AND P. ROUGE
Table 4. Competitive inhibition
Contact
period with
blast, lip No.
of
after
ex30min
ectod. plants Induction
treatment
Induction
frequency
(%)
No.
of
a-D-man.
Oh
4h
24 h
20
56
—
0
53
—
0
94-6
—
24 h
20
52
4
0
48
4
0
92-8
100
(%)
a-D-gal.
(0/20)
(53/56)
—
4
23
4
PSA + a-D-man.
Oh
4h
Induction
frequency
explants Inductior i
0
22
4
0
95-6
100
(0/4)
(22/23)
(4/4)
SBA +a-D-gal.
(0/20)
(48/52)
(4/4)
8
34
4
0
31
4
0
91-2
100
(0/8)
(31/34)
(4/4)
(b) PSA like SBA strongly inhibits neural induction in ectoderm treated
for 30 min and associated for 4 h with blastoporal lip (Table 3 b).
(c) The presence of the inducing tissue for 24 h once more allowed induction
in all treated explants (Table 3 c).
Here again we observed that PSA had no toxic effect. We must underline
that embryonic cells react with pea lectin indicating the presence of complex
N-glycans hitherto detected only in mature tissues.
(C) Competitive inhibition
In order to test the specificity of lectin for its corresponding sugar and to
verify that inhibition of induction results from lectin effects, competitive
inhibition with the sugar required was attempted; SBA specifically binds
a-JV-acetyl-D-galactosamine and a-D-galactose residues; PSA specifically binds
a-D-mannose. A final sugar concentration of 0-1 M (2 %) is sufficient to obtain
maximal saturation of lectin-receptors.
Controls
Sugars (01 M) had no toxic effect on cell viability, no inductive property
on presumptive ectoderm, and did not prevent neural induction occurring in
a normal way.
Competitive inhibition
Results reported in Table 4 indicate that these two lectins, which exert
similar effects, were neutralized when the corresponding sugar was present in
the incubating medium.
Target cell surface in neural induction
183
Each lectin was preincubated (30min) with the suitable inhibiting sugar.
Ectoderm after treatment by lectin + sugar solution was associated with the
inductor for 4 h or 24 h. In these cases, we observed a rate of induction
identical to that obtained for control batches (Table 4).
a-D-galactose for SBA and a-D-mannose for PSA elicited suppression of
the effects of the lectins. Induction rates reached 79/86 (Table 4) whereas the
lectin effects went down to 17/86 (Table 3).
DISCUSSION
For transmission of neural inductive signal in Pleurodeles waltl, 4 h is a
minimum length of time for association between target cells and inducer; this
duration allows neural induction for approximately 90 % of explants. We also
noticed a few positive cases obtained with 215 h association. All experiments
described were performed with 4 h association. Several points were brought
to light:
(1) Lectins are very useful tools for analysing neural induction and differentiation. Nevertheless it is necessary in such an experiment to pay great attention
to the experimental conditions (optimal concentration and duration of treatment) so as not to invoke toxic and unspecific effects. Moreover, Riikola &
Weber (1981) comparing several lectin preparations (PHA), underlined the
presence of contaminating elements and differences in purity between various
batches of the same lectin.
After isolation, we controlled the purity of our preparations by a very
sensitive electrophoretic method. No contamination was noted with polyacrylamide gels.
(2) Ectoderm cells from young gastrulae possess receptors for SBA and
PSA according to other authors. This indicates that surface carbohydrates
contain a-D-galactose and a-D-mannose.
(3) Presumptive ectoderm in vivo exhibits characteristic changes in the cell
surface treated by fluorescent SBA or PSA.
(4) SBA and PSA (50/*gml-\ 30min) have no inducing effect on the
isolated presumptive ectoderm (77 treated explants) cultured for several weeks.
This result is to be compared with the observations of Takata, Yamamoto &
Ozawa (1981) with Concanavalin A (Con A) and Ulex europeus agglutinin
(UEA). These authors, using very high concentrations (100 to 500 /*g ml" 1 for
2 or 3 h), observed an inducing effect by the two lectins, on isolated presumptive
ectoderm in Triturus pyrrhogaster. Other lectins tested (succinyl-Con A, DBA,
WGA, RCA, PNA) had no inducing effect. Con A and UEA seem to be
lectins which differ from the others. However, in these experiments it was not
totally excluded that these agglutinins used at very high doses involved toxicity,
some degenerating cells becoming inducers (cf. Saxen & Toivonen, 1962); the
184
A. M. DUPRAT, L. GUALANDRIS AND P. ROUGE
possible presence of some contaminating elements could not be excluded
either.
(5) The treatment of presumptive ectoderm by SBA or PSA (50 ju,g ml"1,
30 min) previous to association with natural inducing tissue, for 4 h, strongly
inhibited neural induction. During this length of time ectoderm surface conformation is modified (patching and capping of glycoconjugates). The ult/astructural observation of treated ectoderm explants immediately, 4 h and 24 h
after the treatment, shows a normal morphology of all cytoplasmic and nuclear
organelles (mitochondria, Golgi apparatus, reticulum, nuclear membrane,
chromatin, nucleoli, . . . ) .
When the association of previously treated presumptive ectoderm and
blastoporal lip was maintained for a long time: 24 h, neural induction occurred.
As the blastoporal lip remains an inductor during all this time this result
could be explained by the glycoconjugate turn-over which repaired normal
organization of the cell surface (Marcus & Hirsch, 1963; Kraemer, 1976). The
inductive process could then take place (a few hours later than in the control).
We also checked that both SBA-FITC and unlabelled SBA or PSA-F1TC
and unlabelled PSA had similar biological effects on competent target tissue.
Lectins very quickly disturb the structural integrity of the cell surface and
crowd it.
These experiments indicate that the structural integrity of the cell membrane
is necessary for neural induction to occur and suggest that surface glycoconjugates binding to SBA and PSA might be involved in the inductive signal
mechanism. However we do not know if the lectin receptors are directly
concerned in this process. Several hypotheses can be proposed:
(1) The inductive signal requires membrane receptors and lectin receptors,
a-D-galactose and a-D-mannose containing complex, could be directly concerned.
(2) Normal organization of the target cell membrane is necessary for the
transmission of the inductive signal. The lectins involving a disorganization
of the membrane structure (glycoconjugate reorganization), inhibit the inductive
process. What about the relationship between disorganized membrane and
cytoskeleton or internal reticulum? The existence of a relationship between cell
surface receptors and cytoplasmic structural elements has already been demonstrated (Ash & Singer, 1976; Sundquist & Ehrnst, 1976; Albertini & Anderson,
1977; Toh & Hard, 1977).
(3) Lectin molecules bound to membrane sites crowd the cell surface and
prevent the passage of the inductive signal.
Experiments along these lines are now underway to invalidate or corroborate
these hypotheses.
Considering our results together with those reported by others (Tiedemann
& Born, 1978) one may assume that competent target cells are implicated in
the specificity of the neural inductive process; the plasma membrane conformation
plays directly or indirectly an active role, i.e. the structural integrity of the
Target cell surface in neural induction
185
membrane of competent target cells is necessary for neural induction to
occur.
RESUME
Une organisation particuliere des surfaces cellulaires est-elle necessaire au deroulement
de l'induction neurale? Quel role la membrane des cellules-cibles joue-t-elle dans ce processus?
Une cellule embryonnaire traitee par une lectine (glycoproteine ayant une grande affinite
pour un sucre specifique) presente des remaniements de sa surface lies a une redistribution
des glycoconjugues membranaires. Cette propriete a ete utilisee pour perturber et analyser
ainsi le role eventuel de la surface des cellules-cibles competentes, pendant l'induction neurale. Les lectines utilisees sont la SBA et la PSA, fluorescentes ou non.
Chez les Amphibiens, il est possible d'obtenir l'induction neurale in vitro par association
de la levre blastoporale (inducteur) et de 1'ectoblaste (tissu cible). Nous avons etabli avec
exactitude quelle etait la duree de contact necessaire entre le tissu inducteur et le tissu cible
pour que la transmission du signal inducteur ait lieu chez Pleurodeles waltl. Une duree de
contact de 4 h est suffisante, on peut ensuite isoler 1'ectoblaste qui se differenciera en tissu
neural.
Le traitement de 1'ectoblaste par les lectines provoque tres rapidement des modifications de
surface (SBA-FITC et PSA-FITC) et done un remaniement de la structure membranaire.
Les lectines SBA ou PSA aux doses utilisees (50 fi% ml-1, 30 mn) ne sont pas des facteurs
inducteurs.
Traitement prealable de 1'ectoblaste pendant 30', suivi d'un contact de 4 h avec l'inducteur:
pas d'induction.
Traitement identique suivi d'une association avec l'inducteur pendant 24 h: induction
(turnover des glycoconjugues aura reconstitue, tout au moins partiellement, la structure
membranaire).
Une inhibition competitive entre lectine et sucre, prealable au traitement, supprime les
effets de la lectine (SBA/a-D-Galactose; PSA/a-D-Mannose).
Vintegrite structural de la membrane des cellules-cibles est necessaire a Vinduction neurale
et semble etre un element important dans la specificite du phenomene d'induction.
This work was supported by a grant from the C.N.R.S. We thank Dr C. Orfila for kind
hospitality (fluorescence observations), Mrs C. Daguzan for photographic assistance, Mrs
C. Mont for secretarial help and Dr P. Winterton for reviewing the English manusciipt.
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(Received 9 November 198J, revised 25 January 1982)