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.
/ . 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.
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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
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Fig. 1. Purity of PSA(1) and SBA
