ABSTRACT
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 μg ml-1, 30 min) of presumptive ectoderm did not result in any neural induction.
Lectin-treatment (50 μg 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.
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 N-acetylgalactosamine or galactose and Pea lectin (PSA) which has a complex binding requirement that involves N-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 G100 were products of Pharmacia, Uppsala, Sweden. l-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HC1 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 Coo-massie 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 Sepharose-N-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 g for 20 min. The supernatant was applied to a column of Sepharose-7V-caproylgalactosamine (5 ×1 ·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 0 ·05 at 280 nm. Lectin was then 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 0 ·15M-NaCl, 0 ·05 MTris-HCl buffer pH 7 ·6 (250 ml) and the mixture was centrifuged at 15000 g 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 (70 ×2 ·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 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 (pH 9 ·5) containing 10 mg of lectin was incubated at 25 °C and 250 μl of phosphate buffer containing 200 μg 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-A-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 presumptive ectoderm and the blastoporal lip were microsurgically excised in Holtfreter solution including penicillin (100 i.u. ml-1), streptomycin (100 μg 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 :
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 μ m), stained by the Unna technique and examined histologically.
Ectoderm was dissociated into a single cell suspension with Barth dissociation medium (NaCl, 88 mM; KC1, 1 mM; NaHCO3, 2 ·4 mM; Na2HPO4, 2 ·0 mM; KH2PO4, 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, 100 μg ml-1 were studied. Optimal concentrations were found to be 25 or 50 μg 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 expiants 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) between SBA/ α-D-galactose 0 ·1M (2%) and PSA/ α-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 expiants 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.
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.
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).
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).
(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. 4b). These patterns were to be observed for several hours. This process 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.
Presumptive ectoderm treated with fluorescent SBA (50 μg ml –1, 20 °C, 5 min), (a) First, fluorescent line appears only around the cells; (b) Patches and caps appeared after, indicating surface modifications.
(B) Effects on neural induction
Table 2 summarizes the experiments performed.
(i) Soybean lectin
In ectoderm explants treated with SBA (50 μg 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).
- Ectoderm explants treated with SBA (50 μg 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.Fig. 5.
Ectoderm treated with SBA (50 μgml –1) associated with inductor tissue 4 h: neural induction is inhibited, only epidermal differentiation is observed in cell culture.
- Ectoderm treated with SBA (50 μg 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).Fig. 6.
Ectoderm treated with SBA (50 μg ml –1) associated with inductor tissue 24 h : neural induction occurs normally (cell culture).
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 μg 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).
The control experiment showed the absence of neuralizing effect on competent presumptive ectoderm (Table 3 a).
PSA like SBA strongly inhibits neural induction in ectoderm treated for 30 min and associated for 4 h with blastoporal lip (Table 3 b).
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 α-N-acetyl-D-galactosamine and α-D-galactose residues; PSA specifically binds α-D-mannose. A final sugar concentration of 0 ·1 M (2 %) is sufficient to obtain maximal saturation of lectin-receptors.
Controls
Sugars (0 ·1 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.
Each lectin was preincubated (30 min) with the suitable inhibiting sugar. Ectoderm after treatment by lectin + sugar solution was associated with the inductor for 4h or 24 h. In these cases, we observed a rate of induction identical to that obtained for control batches (Table 4).
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 2 ·15 h association. All experiments described were performed with 4 h association. Several points were brought to light :
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.
Ectoderm cells from young gastrulae possess receptors for SBA and PSA according to other authors. This indicates that surface carbohydrates contain α-D-galactose and α-D-mannose.
Presumptive ectoderm in vivo exhibits characteristic changes in the cell surface treated by fluorescent SBA or PSA.
SBA and PSA (50 μg ml –1, 30 min) 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 μg 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 possible presence of some contaminating elements could not be excluded either.
The treatment of presumptive ectoderm by SBA or PSA (50 μg 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 ultrastructural 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 :
The inductive signal requires membrane receptors and lectin receptors, α-D-galactose and α-D-mannose containing complex, could be directly concerned.
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).
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 membrane of competent target cells is necessary for neural induction to occur.
RÉSUMÉ
Une organisation particulière des surfaces cellulaires est-elle nécessaire au déroulement de l’induction neurale? Quel rôle la membrane des cellules-cibles joue-t-elle dans ce processus?
Une cellule embryonnaire traitée par une lectine (glycoprotéine ayant une grande affinité pour un sucre spécifique) présente des remaniements de sa surface liés à une redistribution des glycoconjugués membranaires. Cette propriété a été utilisée pour perturber et analyser ainsi le rôle éventuel de la surface des cellules-cibles compétentes, pendant l’induction neurale. Les lectines utilisées 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 lèvre blastoporale (inducteur) et de l’ectoblaste (tissu cible). Nous avons établi avec exactitude quelle était la durée de contact nécessaire entre le tissu inducteur et le tissu cible pour que la transmission du signal inducteur ait lieu chez Pleurodeles waltl. Une durée de contact de 4 h est suffisante, on peut ensuite isoler l’ectoblaste qui se différenciera en tissu neural.
Le traitement de l’ectoblaste par les lectines provoque très rapidement des modifications de surface (SBA-FITC et PSA-FITC) et donc un remaniement de la structure membranaire.
Les lectines SBA ou PSA aux doses utilisées (50 μg ml –1, 30 mn) ne sont pas des facteurs inducteurs.
Traitement préalable de l’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 glycoconjugués aura reconstitué, tout au moins partiellement, la structure membranaire).
Une inhibition compétitive entre lectine et sucre, préalable au traitement, supprime les effets de la lectine (SBA/ α-D-Galactose; PSA/ α-D-Mannose).
L’intégrité structurale de la membrane des cellules-cibles est nécessaire à l’induction neurale et semble être un élément important dans la spécificité du phénomène d’induction.
ACKNOWLEDGEMENTS
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.