J. Embryol. exp. Morph. 77, 183-200 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
Membrane changes in neural target cells studied
with fluorescent lectin probes
By L. GUALANDRIS 1 , P. ROUGE 2 AND A. M. DUPRAT 1
From the Laboratoires de Biologie generate (ERA-CNRS n°327) et de
Biologie cellulaire (Faculte de Sci. Pharm.), Universite Paul Sabatier
SUMMARY
The competent ectoderm of Pleurodeles waltl comprises two cell layers with characteristic
differences in their morphology, their composition and the molecular arrangement of the
various constituents.
The use of labelled lectin probes for observations of ectoderm tissue in vitro with u.v.
microscopy (epi-illumination) and the quantification of the results show the following:1) Differences in labelling according to the nature of the lectins (SB A, PSA, LCA and Con
A). These differences provide information on the nature of the carbohydrates which are
present at this stage and on the number of receptors.
2) Differences in fluorescence intensity of the surfaces studied. The internal surface of the
ectoderm is labelled more densely than the external surface.
3) Rearrangement of the lectin receptors with a new molecular configuration, stressing the
fluidity of the membrane (by the mobility of the receptors throughout the membrane) and its
importance for the occurrence of neural induction.
4) Existence of membrane glycoconjugate turnover.
5) A difference in behavioural characteristics between the internal and the external surfaces with respect to the lectins and the formation of an extracellular matrix on the internal
surface alone. The extracellular matrix seems to have a role in morphogenetic movements.
INTRODUCTION
It has been shown that the presumptive ectoderm cells of gastrulae exhibit
characteristic changes during development (Tarin, 1971; Monroy, Baccetti &
Denis-donini, 1976; Kohonen & Paranko, 1978; Grunz & Staubach, 1979). As
we have already reported (Duprat, Gualandris & Rouge, 1982), the molecular
organization of the plasma membrane of the target cell seems to play a crucial
role at the gastrula stage in the process of neural induction. The plasma membrane must possess a special organization and possibly the process of induction
itself results from special molecular reorganizations in the same membrane
(Takata, Yamamoto & Ozawa, 1981).
1
Authors' address: Laboratoire de Biologie g6nerale, Universite Paul Sabatier, 118 route
de Narbonne, 31062 Toulouse Cedex, France.
2
Author's address: Laboratoire de Biologie cellulaire, Universit6 Paul Sabatier, 118 route
de Narbonne, 31062 Toulouse Cedex, France.
184
L. GUALANDRIS, P. ROUGE AND A. M. DUPRAT
Therefore since many important developmental phenomena must operate
through cell surfaces, the aim of this work is to study certain cell surface properties of the responding tissues in neural determination.
Lectins are a group of proteins (essentially glycoproteins) which are able to
bind specifically to carbohydrate moieties on the cell surface. Such marker
probes have been employed in amphibians (Johnson & Smith, 1976; Nosek,
1978; Tencer, 1978; Boucaut et al. 1979; Barbieri, Sanchez & Delpino, 1980;
Takata, Yamamoto & Ozawa, 1981) using fluorescent-labelled lectins to
visualize and study the dynamics of cell surface glycoconjugates in morphogenesis.
We have previously reported the important roles played by cell surface glycoconjugates and their topography in the process of neural induction. The present
experiments were designed to elucidate the nature and turnover of the glycoconjugate molecules present on target cell surfaces of Pleurodeles waltl, which might
be involved in this process. We used fluorescent-labelled lectins which were
specific for N-AC-a-D-galactosamine, a-D-galactose (SBA); a-D-mannose and
a-D-glucose (PSA, LCA and Con A).
MATERIALS AND METHODS
Pleurodeles waltl gastrulae (stage 8) were used as donors of presumptive
ectoderm and were staged according to the table of development established by
Gallien & Durocher (1957).
After removal of the jelly coat and the vitelline membrane, the ectoderm
(target tissue) was microsurgically excised in Holtfreter solution including
penicillin (lOOi.u. ml"1); streptomycin (lOO/igml"1) and buffered with 5mMTris;pH8-0.
These ectoblast explants were incubated for various lengths of time (1, 3, 15
& 30 min) at 20 °C in Holtfreter medium containing lectin (50 /ig ml"1) (Gualandris&Duprat, 1981 ;Dupr at etal. 1982). They were then washed thoroughly with
Holtfreter solution and observed in vitro.
1. Preparation of lectins
CH-Sepharose 4B (which is formed by covalent linkage of 6-amino hexanoic
acid to CNBr-activated Sepharose 4B) and Sephadex G100 were products of
Pharmacia, Uppsala, Sweden; l-ethyl-3-(3-dimethylaminopropyl) carbodiimideHCL 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, T.E.M.E.D. and
Coomassie brilliant blue R were from BioRad, Richmond, U.S.A. Ultragel
ACA202, di-chlorotriazinyl-fluorescein (D.T.A.F.) and tetramethyl-rhodaminisothiocyanate (TRITC) were supplied by IBF, Villeneuve-la-Garenne, France.
N, N-dimethyl-formamide was purchased from Fluka, Buchs, Switzerland, and
Membrane changes studied with fluorescent lectin probes
185
Jack-bean meal from Serva, Heidelberg West Germany. All other reagents were
commercial preparations of Merck (pro analysis grade).
Soy-bean lectin: SBA
Soy-bean lectin (SBA) was purified by affinity chromatography on SepharoseN-caproylgalactosamine prepared according to Allen & Neuberger (1975), and
frozen at -30°C until analysis but for no more than 2 months.
Pisum sativum lectin: PSA
Garden-pea lectin (PSA) was isolated by affinity chromatography on
Sephadex G100 (Agrawal & Goldstein, 1967) and then stored frozen at below
- 3 0 °C.
These methods have been published (Duprat et at. 1982).
Lens culinaris lectin: LCA
Preparation as for PSA, but with lentil meal.
Canavalia ensiformis lectin: Con A
Preparation as for PSA, but with Jack-bean meal.
2. Purity of lectins
The purity of the SBA, PSA, LCA and Con A 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).
The protein content of the SBA, PSA, LCA and Con A preparations was
estimated by the microbiuret procedure of Goa (1953) using a bovine serum
albumin standard.
3. Lectin labelling
As previously described (Duprat et al. 1982), fluorescent-labelled lectins
(FITC-SBA, FITC-PSA, FITC-LCA) were prepared according to the slightly
modified procedure of The & Feltkamp (1970).
Labelling of SBA, PSA and LCA by TRITC was performed under the same
conditions as FITC labelling, but using a fluorochrome solution of twice the
concentration, since TRITC labelling is less effective than FITC labelling. However, labelling of Con A was conducted using a modified procedure, DTAF
instead of FITC was used. TRITC or DTAF were dissolved in N, N-dimethylformamide prior to coupling; 40 /ig of TRITC or 20/^g of DTAF per mg of
protein were added to lectin solubilized in 0-1 M-carbonate buffer pH9-0 under
continuous stirring. The reaction was continued in the dark, at 4°C for 4h. The
186
L. GUALANDRIS, P. ROUGE AND A. M. DUPRAT
1
LCA
PSA
SBA
Con-A
Fig. 1. Purity of lectins by polyacrylamide gel electrophoresis: At alkaline pH PSA
shows two main bands corresponding to the isolectins previously described (Entlicher, Kostir & Kocourek, 1970). In the same conditions SBA also shows two bands
while at acid pH only a single band is obtained. Clearly 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). Con A gives a single band under
the same conditions, however gels show Con A with probably degradation products
(Liener, 1976). LCA, which comprises two isolectines (Howard & Sage, 1969) gives
two closely related bands on electrophoresis.
labelled lectins were separated from free DTAF or TRITC by filtration through
a column of Ultra gel ACA202 using 0-05M-Tris, (M5M-NaCl pH7-6 as eluent.
Fig. 2A, 2B, illustrate these two described methods to separate labelled lectins
from free fluorochromes.
The labelled lectins were subsequently concentrated by precipitation with
70 % saturated ammonium sulphate. After extensive dialysis against 0-05 M-Tris,
0-15M-NaCl pH7-6 the lectin solutions were stored frozen and always used
within two months in order to prevent any possible molecular aggregation or
release of free fluorochrome.
As in the case of unlabelled lectins, the purity of the labelled lectins was
checked by polyacrylamide gel electrophoresis and result was identical to that
shown Fig. 1.
4. Fluorescence microscopy
A Leitz Dialux epifluorescence microscope with an incident source HBO 50
Membrane changes studied withfluorescentlectin probes
20
187
2A
00
Fractions
2-0
2B
00
Fractions
Fig. 2A. Isolation of TRITC-labelled by affinity chromatography on Sephadex G 75
column. Arrow 1: 0-05M-Tris, 0-15M-NaCl pH7-6 buffer. Arrow 2: identical
buffer + 0-1 M-glucose. The first peak corresponds to free TRITC and the second to
the labelled LCA released by addition of glucose to the elution buffer, the same
pattern is observed for FITC-LCA, FITC-PSA and TRITC-PSA.
Fig. 2B. Isolation of FITC-labelled SBA by chromatography on a ACA 202 column.
1st peak: labelled lectin. 2nd peak: free fluorochrome. The same pattern is observed
for TRITC-Con A, TRITC-SBA and DTAF-Con A.
orthomat with filter I2 (BP 450-490, LP 515), was used to examine cells for FITC
labelling and with filter M2 (BP 546/14., LP580) in order to examine cells for
TRITC labelling. Fluorescence microcraphs were made on Ektachrome
160 ASA film with a Wild MPS45 camera.
188
L. GUALANDRIS, P. ROUGE AND A. M. DUPRAT
5. Fluorescent lectin inhibition
The binding of each fluorescent lectin was prevented when its inhibitory sugar
was co-present in the incubation solution, or when the labelling lectin was preincubated in the suitable inhibiting sugar, at the concentration of 0-1 M (SBA,
a-D-gal; PSA, LCA and Con A, a-D-man).
6. Quantitative results of fluorescence
Quantification of the fluorescence intensity was performed with a Leitz
microscope coupled to an MP V2 type photomultiplier. The measurements in
Table 1 were made at 0-6 kV (S = 1) and at 0-5 kV (S = 1) in Table 2.
The explant was scanned through a window of 178 jinn diameter (covering 18
to 20 cells at a time) and the fluorescence of each explant surface was measured
at 20 to 25 spots so as to cover its whole area. Each experiment used at least three
explants. These series of measurement supplied a set of intensity values (arbitrary units) from which the means were determined.
The test of Kolmogorov-Smirnov adapted for the study of two samples of nonnormal distribution was used to interpret the results (Siegel, 1956).
RESULTS
In situ, the presumptive ectoderm of P. waltl consists of a double cell layer
(Fig. 3). Intact ectoderms were used immediately after their dissection from
gastrulae and observation with bright-field microscope revealed that the external
side comprises small highly pigmented polyhedral cells (Fig. 4), while the internal side is formed of larger spherical cells and is only very slightly pigmented
(Fig. 5).
I. Reorganization of lectin receptors
1. Control observations
Explants not treated by fluorescent lectins did not show any autofluorescence.
Lectin binding is rapid: explants exhibit fluorescence after an incubation of
1 min. The same pattern of fluorescence was also found when the explants were
treated for longer times (3 min or 15 min). Whether the lectins were labelled with
Fig. 3. Histological section (7-5 ^m; U.N.N.A. colouration) of presumptive
ectoderm of gastrula (stage 8a) of Pleurodeles waltl. Two cell layers are visible.
Bar= 10 pim.
Fig. 4. External surface of presumptive ectoderm of early gastrula stage (stage 8a).
The cells of this layer are small and polyhedral with strong pigmentation. Bar =
Fig. 5. Internal surface of presumptive ectoderm of early gastrula stage (8a). The
cells are round and slightly pigmented. Bar =
Membrane changes studied withfluorescentlectin probes
7
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Figs 3-5
EM$77
189
190
L. GUALANDRIS, P. ROUGE AND A. M. DUPRAT
FITC or TRITC their behaviour with respect to the explants was identical. In our
experimental conditions lectin binding was stable, being still observed several
hours after fixation. Lectins used at the concentration SO/igml"1 did not exert
toxic effects on the explants: treated cells lived as long as the controls, and no
nuclear or cytoplasmic anomalies or cytolysis or abnormal behaviour were observed in these cells (observations on living cells for 3 or 4 weeks and ultrastructural
studies).
2. Fixation of lectins on the two explant surfaces
Ectoderm explants were treated in vitro with fluorescent lectins in order to
check the binding of the lectins at the cell surface, to investigate the modifications brought about in the membrane topography of cells aggregated in a tissue
explant and to compare the behaviour of the outer and the inner surfaces of the
target tissue.
After lectin binding a reorganization of the topographical structure of the
membrane takes place with a rearrangement of the various receptor sites on both
surfaces of the explant. The ectoderm explants differ morphologically on their
internal and external surfaces.
External surface. Lectin binding results in a wide fluorescent band at the
periphery of cells. The fluorescent moieties are evenly spread over the cells (Fig.
6A) but are apparently restricted to spaces between cells. After binding, the
ectoderm explants show a rearrangement of bound lectins. The redistribution of
fluorescent-lectin binding results in small patches, which are rapidly brought
together at one pole of the cell in the form of a cap (Fig. 6B, 6C).
This aggregation of fluorescence from the diffuse band on the cell periphery
into clusters of various sizes, is rapid. Lectin binding is almost instantaneous and
receptor migration occurs in less than 15min. In some cases the fluorescence
pattern shows caps formed in less than 1 min.
Internal surface. First, there is fluorescence in a thin intense band at the
periphery of the cells (Fig. 7A). Then, the fluorescence develops into a fine
network on each cell with a spoke-like structure which seems to converge at a
central point (Fig. 7B, 7C). Just above the level of the cell surface a large
spreading network of tangled filaments which densely bind lectins is formed (Fig.
8). This filamentous material remains stuck to the support when the cells are
detached, indicating its extracellular location.
Since, it is the inner surface of the ectoderm which undergoes neural induction
in the embryo, could this extracellular matrix play a role in cell induction? No
network of extracellular material was ever observed on the outer surface of the
Fig. 6. External surface of ectoderm incubated in FITC-SBA for 3min. (A) immediate observation: homogeneous distribution of lectin around the cells. (B) after
2-3 min, each cell presents several clusters of bound FITC-SBA. (C) after 10 min,
capping phenomenon indicating surface modifications. Bar =
Membrane changes studied withfluorescentlectin probes
B
Fig 6A-C
191
192
L. GUALANDRIS, P. ROUGE AND A. M. DUPRAT
if;
Fig 7A-C
Membrane changes studied withfluorescentlectin probes
193
Fig. 8. Inner surface of ectoderm incubated in FITC-SBA. Just above the level of
the cell surfaces, a large spreading network of filamentous material is observed.
Bar = 10|Um.
ectoderm. To clarify this point we associated in vitro the natural inductor tissue
(blastoporal lip) onto the outer surface of the isolated ectoderm according to the
methodology previously described (Gualandris & Duprat, 1981). Induction
occurred in this experiment (25/25 positive cases) as for a blastoporal lip association onto the inner surface of the explant. We concluded that the extracellular
matrix on the inner surface of the ectoderm does not play an essential role in
neural determination.
Thus our experiments indicate that 1) the capping phenomenon, only
described on isolated cells, can also occur when the cells are aggregated in a
tissue; 2) the molecular reorganization with respect to lectin binding is different
for the external and the internal sides of the ectoderm.
Table 1 shows comparative studies of the fluorescence intensity with the different lectins (SBA, PSA, LCA and Con A) on the external and the internal
surfaces of ectoderms.
In all cases 1) the lectin is rapidly bound and the fluorescent patterns differ as
described above, but each surface acts in the same way towards the various
lectins; 2) the lectin binds more densely to the inner than to the outer surface.
The experiments also pointed out that the order of intensity of fluorescence is:
Con A < LCA < PSA < SB A. Moreover the ratio of external side/internal side
fluorescence is constant (=1/2).
Fig. 7. Internal surface of ectoderm incubated in FITC-SBA for 3min. (A) thin
fluorescent line at the periphery of the cells. Bar = 20fjm. (B and C) then, on each
cell, a spoke-like structure seems to converge to a central point on the cell. (B)
Bar = 20 jum. (C) Bar = 10 jum.
194
L. GUALANDRIS, P. ROUGE AND A. M. DUPRAT
Table 1. Comparative study of the fluorescence intensity (in arbitrary units), with
different lectins, of the internal and the external surfaces of treated ectoblast
Lectins
SBA
PSA
LCA
CON A
Fluorescence of
lectins in
solution
(SOjUgml"1)
No.
Fluorescence of
the external
surface
No.
Fluorescence of
the internal
surface
No.
100-52 ±0-398
*100
20
46-92 ±1-928
*45 76±191
15
96-84 ±3-090
*96 33 ± 3 0 7
15
20-99 ± 0-436
*100
52-02 ± 0-890
*100
11
6-54 ±0-295
*31 15 ± 1 4 0
33
11-83 ±1-485
*56 36 ±7 07
39
10
6-74 ± 0-053
*12 95±0 10
20
11-44 ±0-161
*21 99 ± 0 31
30
54-64 ± 0-810
10
*" 2-26 ±0-010
34
4-215 ±0-495
55
*100
*4 13
*7-71 +0-90
No. = Numbers of measurements.
* = The actual values observed are in roman print; bold print corresponds to values expressed as a "100" of the fluorescence of the lectin solution used.
The changes in the fluorescence pattern described above occur rapidly (in less
than 15 min at 20 °C) and the pattern is not further modified with time. It is still
present several hours after lectin binding, indicating that, under our experimental conditions, the binding of lectins to the cell receptors is not labile. It should
however be noted that the intensity of fluorescence decreases with time (4 h, 6 h,
24 h). This phenomenon will be discussed in relation to glycoconjugate turnover.
II. Turnover of membrane glycoconjugates
Experiments using labelled SBA, PSA and LCA, were performed to observe
the kinetics of changes in the explants, in vitro, over 24 h and more. The different
studied explants were placed in the same conditions of temperature, pH and
darkness.
Experiment 1. When the explants, incubated in unlabelled lectin for 3 min, were
incubated in fluorescent lectin: no fluorescence was observed on the explant
surfaces. All the lectin-binding sites were saturated with the first incubation.
Experiment 2. If the explants were incubated in lectin solution containing the
corresponding inhibitory sugar (SBA: a -D-galactose, PSA: a-D-mannose) no
fluorescence was observed. This indicates that the reaction was specific because
the fluorescence was abolished in the presence of the inhibiting sugar. The
fluorescence normally observed results in specific binding of labelled lectins.
Experiment 3. Immediately after incubation in the fluorescent lectin solution
Membrane changes studied withfluorescentlectin probes
195
Table 2. Quantitative analysis of thefluorescenceintensity against time, on treated
ectoblast.
- FITC-SBA treatment Fluorescence values
(mean ± S.D.)
No.
(Number of measurements)
23-99 ±0-102
25-29 ±0-293
19
44
B) Experiment 3
3 min treatment
5-01 ±0-143
69
C) Experiment 4
3 min treatment +
24 h normal medium
2-89 ± 0-052
70
9-30 ±0-143
72
19-22 ± 0-533
73
Treatment
A) Lectin solution
. Immediate observation
. Observ. after 24 h
D) Experiment 5
3 min treatment +
24 h normal medium +
3 min treatment
E) Experiment 6
24 h treatment
The Kolmogorov-Smirnov test pointed out significant differences between all these experiments at P< 0-01 (see Results).
Similar results were obtained with PSA and LCA.
(3 min) the explants were washed and examined: fluorescence was intense (Table
2B).
Experiment 4. When explants were treated in the same way as for experiment
3 and then placed in normal Holtfreter medium for 24 h, thefluorescenceintensity
was seen to decrease but not to totally disappear; glycoconjugate reorganization
was still observed (Table 2C).
Experiment 5. Similar explants, incubated in fluorescent lectin for 3 min then
placed for 24 h in normal medium, were incubated a second time in the fluorescent lectin solution for 3 min: the fluorescent intensity was high (Table 2D).
Experiment 6. Explants continually incubated for 24 h in fluorescent lectin
solution presented a very high fluorescence intensity, probably resulting from the
addition of the normal fluorescence to the accumulation of released material
(Table 2E).
Under our experimental conditions the labelled lectins were unaltered after
24 h storage, and the fluorochromes did not lose any of their activity (Table 2A).
196
L. GUALANDRIS, P. ROUGE AND A. M. DUPRAT
In experiment 4, the low fluorescence reflects the disappearance of lectin-binding
receptors. In experiment 5, the fluorescence is again intense; this seems to result
from the renewal of the receptors. The new receptors then bind the lectin when
it is introduced into the culture medium.
DISCUSSION
It is now well established that lectin binding to the cell surface brings about a
rearrangement of the membrane constituents which bind the lectin (Inbar &
Sachs, 1973;Nicolson, 1973,1974,1976; Garridoetal. 1974; Zalik&Cook, 1976;
Sharon, 1977). The use of lectins conjugated with fluorochromes demonstrated
the presence of lectin receptors at the surface of Pleurodeles cells. It enabled their
nature to be determined and their mobility in the membrane to be analysed.
We took the precaution of isolating, purifying and checking the labelling of all
the lectins used. Great variations were noticed in commercially available batches
of lectins with lack of homogeneity and presence of contaminating elements
(Riikola & Weber, 1981,1982). It appears, that our lectin batches were of good
quality, giving no artifactual effects on the materials studied.
Comparative studies of internal and external surfaces of the ectoderm by
direct labelling of the explants were carried out. In this way the fluorescent
lectins show that lectins bind more densely to the inner surface. Moreover the
differences in lectin binding between the two surfaces indicates that the inner
surface possesses receptors for SB A and PSA as well as for LCA and Con A,
implying that the surface glycoconjugates include N-Ac-a-D-galactosamine,
a-D-galactose, a-D-mannose and a-D-glucose receptors.
The order of binding intensity on the outer surface was SBA > PSA > LCA
> Con A; but Con A was not always found in detectable amounts. So, the outer
surface seems to possess receptors of the a-D-galactose type, the others being
less frequent or less accessible to the lectins. The results obtained with PSA and
LCA, both of which bind preferentially to the same sugar: a-D-mannose, suggest
a difference in their affinity for their receptors. Indeed the fluorescence observed
with PSA was more intense than that obtained with LCA even though the number of moles of fluorochrome bound per mole of lectin was identical.
It is interesting to point out that the embryonic cells react with pea lectin,
indicating the presence on the cells of N-glycan complexes (N-Ac-glucosamine,
mannose, fucose receptors) which until now had only been detected in adult
tissue (Kornfeld, Reitman & Kornfeld, 1981).
Direct labelling would seem to indicate the absence of certain lectin receptors
at the outer cell surface of the explants. Several comments can be made: either
the carbohydrates are indeed absent, or they are present but not accessible, or
not visualized. As far as the availability of the receptor sites is concerned,
Moscona (1971) showed that the receptors of certain lectins are present at the
surface of embryonic cells in a masked form.
Membrane changes studied with fluorescent lectin probes
197
Our results are in agreement with those obtained in Xenopus laevis using
various lectins (WGA, SB A, RCA, PSA, Con A) on chemically fixed embryos
(Nosek, 1978). The experiments bring to light differences in lectin binding between the internal and the external surfaces especially in the ectoderm layer at
the animal pole of the gastrula. But the PSA and Con A receptors do not seem
to be as numerous in Pleurodeles waltl as in Xenopus laevis.
The fact that we observed a variation in the time of appearance of stable
membrane reorganization patterns can be explained by differences between eggs
in a same batch and/or differences in the exact age of the gastrula studied (stage
8a, 8b). Johnson & Smith (1977) working with Xenopus laevis showed that the
rate of binding-site redistribution varies according to the exact stage of development of the gastrulae; a rise in the number of clusters or caps occurs with increasing age of the gastrulae.
On the inner surface we report two major observations. First, binding of
lectins into the membrane demonstrating the presence of glycoconjugates and
their relative mobility. The fluorescence patterns on each cell develop to form
a spoke-like structure. Second, a filamentous network is formed which could be
composed of extracellular material. This fibrous layer lies over the cells and can
be separated from them; this material remains stuck to the support when the cells
are detached.
The extracellular matrix, first reported in vivo in amphibians by Tarin (1971)
at the interface between the chordamesoderm and the ectoderm during gastrulation, was studied by Johnson (1977) who showed the presence of extracellular
materials that stain with toluidine blue and lanthanum nitrate. Similarly, Kosher
& Searles (1973) found that mucopolysaccharide sulphates were synthesized and
accumulated at the cell surface during gastrulation. Recently, in Ambystoma
maculatum gastrulae, Nakatsuji, Gould & Johnson (1982) described a network
of fine extracellular fibrils that covers the inner surface of the ectodermal layer
and seems to serve as a guiding substratum for migrating mesodermal cells.
Boucaut & Darribere (1983) in Pleurodeles, Bride (1982) in Xenopus and Nakatsuji & Johnson (1983fl,fr) demonstrated the presence of fibronectins at the
chordamesoderm-ectoderm contact during invagination of the blastoporal lip at
the gastrula stage.
CONCLUSIONS
So, using fluorescent lectin probes, it was observed that (1) the inner and the
outer surfaces of the ectoderm present different behavioural characteristics; (2)
the extracellular matrix we observed on the inner surface of the ectoderm (containing N-Ac-a-D-galactosamine, a-D-galactose, a-D-mannose, a-D-glucose
glycoconjugate molecules) does not seem to play an essential role in neural
induction. This matrix is thought to be involved in the morphogenetic movements of gastrulation.
198
L. GUALANDRIS, P. ROUGE AND A. M. DUPRAT
Experiments are currently underway to attempt to accurately produce a time
course for glycoconjugate turnover in Pleurodeles and to assess the role of the
target cell plasmalemma structure in the neural induction process both in vivo
and in vitro.
This work was supported by grants from the CNRS. The authors are indebted to Dr F.
Gassett and Dr G. Farre for the expert assistance in fluorescence quantification, to Mrs C.
Daguzan for photographic assistance, Mrs C. Mont for secretarial help and to Dr S. Jarman
for reviewing the English manuscript.
RESUME
L'ectoblaste competent de Pleurodeles waltl est constitue de deux couches de
cellules qui presentent des caracteristiques differentes dans leur morphologie,
leur composition et l'arrangement moleculaire des differents constituants.
Des lectines couplees a des fluorochromes (FITC ou TRITC) ont ete utilisees
in vivo et l'intensite de fluorescence quantifiee. Ces observations montrent qu'il
existe:
1) des differences dans le marquage des explants selon la nature des lectines
etudiees (SBA, PSA, LCA ou Con A). Ces differences fournissent des indications sur la nature des glucides presents a ce stade et leur quantite.
2) des differences d'intensite de fluorescence selon la face externe ou interne
de l'explant; la surface interne etant toujours plus intensement fluorescente que
l'externe;
3) un remaniement des recepteurs lectiniques avec mise en place d'une configuration moleculaire nouvelle, mettant en evidence une certaine fluidite de la
membrane. Ceci est a relier avec des resultats precedents (JEEM, 70,171-187,
1982) et montre l'importance de la membrane des cellules ectoblastiques dans le
deroulement de l'induction neurogene.
4) un renouvellement des glycocon jugues membranaires;
5) des differences comportementales des deux faces de l'explant, avec la
formation d'une matrice extra-cellulaire uniquement sur la face interne. Cette
matrice n'est pas necessaire au deroulement de l'induction neurogene et semble
done etre impliquee dans les mouvements morphogenetiques de la gastrulation.
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(Accepted 23 May 1983)