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 (α-D-galactose, N-acetyl-D-galactosamine, α-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 ionic fluxes and physicochemical and electrophysiological properties of the target membrane could play a crucial role in neural induction.
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 et al. 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; Barbieri, Sanchez & Delpino, 1980; Gualandris et al. 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 et al. 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 (α-D-mannose, α-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
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 (100 i.u.ml−1) and streptomycin (100 μgml−1). Gastrulae were manually de jellied and the vitelline membrane removed. The microsurgically excised ectoderm was treated with different lectin solutions (50 μg ml−1 or 300 μg ml−1) for 30 min or 3 h. The expiants 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 mM-KH2 PO4, 0·5 DIM 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).
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 04 M-phosphate-buffered saline (pH 7·2) on standard microtitration plates. To each lectin solution (50 μl) were added 200/11 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 50/11. To each sugar dilution 50 μl of PBS containing 50 μg ml−1 of native or succinylated lectin were added. After a 1 h incubation, 200 μl 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 α-D-mannosyl and α-D-glucosyl residues, α-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 (α-D-mannose) in order to prevent their haemagglutinin activity. For a preincubation, 50 μg or 300 μg of lectin was used in 1ml of α 0·1M solution of 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 lectin-binding sites.
None of the biological effects of lectins were observed when the hapten inhibitor was copresent in the solution.
(3) Saturation of binding sites (50 μg ml−1 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.
Expiants were first incubated in a solution of unlabelled lectin (Con A for example) 50 μg ml−1 for 30 min, thoroughly washed and incubated in a solution of the fluorescent lectin (Con A-FITC or TRITC) 50/zgml-1 for 30 min. After several washings the control of the fluorescence of expiants 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.
No fluorescence was detected on the expiants. All the lectin binding sites were saturated by the first incubation. Con A, S-Con A, PsA and LcA (50 μgml−1 for 30min) 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 (300 μgml−1 for 15 or 30min).
Expiants 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:
In all cases, these double-labelling experiments, showed no fluorescence on the treated expiants, 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 300 μgml−1) was carefully checked using exclusion test with trypan-blue dye, ultrastructural cytology, cell behaviour and differentiation over a 10-day period in vitro.
The exclusion test with trypan blue indicated a similar % of dead cells between treated and control batches(<5%)exceptfora300 μgml−1 Con A treatment (⩽10%). 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.
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).
(1) Effects of lectins on isolated presumptive ectoderm (stage 8)
(A) Lectin-treatments for 3 h, 50 μg 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 50 μgml−1 for 3 h, none of the studied lectins had neural inducing properties.
(B) Lectin treatments for 3 h, 300 μg 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 μgml−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 et al. (1981, 1984). .
(b) No ectoderms treated with S-Con A were induced, only epidermal differentiation was observed. Thus S-Con A did not exhibit a neuralizing effect.
(c) (d) No neuralization 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 μg ml−1) prior to association for 4h 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.
To summarize these experiments, the effects of four lectins with affinity for the same carbohydrate residues were studied under saturating conditions:
PsA and LcA were not found to have neural inducing properties (50 μg ml−1 and 300 μg ml−1). Moreover as previously reported they reversibly inhibited the process of induction by the natural inducing tissue (Gualandris et al. 1983).
Con A was not a neural inducer at 50 μg 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 μg ml−1) it had inducing properties.
S-Con A like PsA and LcA did not present an inducing effect (50 μg and 300 μg ml−1). It did not inhibit the natural inductive process.
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 μg ml−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 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 μg 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: α-D-galactose, N-acetyl-α-D-galactosamine, α-D-mannose, α-D-glucose, α-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 Rana pipiens gastrulae, Barth (1965, 1966), Barth and Barth (1967, 1968 and 1974) proposed the following scheme for neural induction: - ‘during gastrulation, ion (Na+, K+, Ca2+, 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 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.