Cell Differentiation. 18 (1986)57-64 57 El~vier ScientificPublishers Ireland, Ltd. CDF 00344 Neural induction and in vitro initial expression of neurofilament and tetanus toxin binding site molecules in amphibians A.M. Duprat i L. Gualandris i, F. Foulquier o D. Paulin 2 and B. Bizzini ~ o umtb associbe.CNRS 675. Laboratoire de Biologw g~nbrale. Unwersirb P. SabatWr. 31062 Toulouse ('edex. : Unitb de Gbnblique Cellulmre and "~Uml~ d'lmmunrs'himie des protbines. Institul Pasteur. 75724 Pari.~ Cedex. Fram'e (Accepted 5 August 1985) Tetanus toxin (Tt) binding site and neurofilament (NIF), the intermediate-sized filaments, are neuronal markers e~ntially described in mammals and birds; are these molecular markers present in urodela neuronal cells and are they expressed immediately after neural induction? Our findings are based on immunofluores- cent localization of NIF and Tt proteins using three previously characterized antisera against 200 kDa and 70 kDa neurofilament components and against fragment Ilc derived from purified tetanus toxin. Embryonic undifferentiated neuronal cells from Pleurodeles ~ l t l i i neural plate and/or neural fold (early neurula stage) are cultured isolated in vitro without further chordamesodermal influence. At the beginning of the culture none of the undifferentiated neuronal precursors bind antibodies against NIF or Tt components. The binding is detected when phenotypical differentiation takes place (2/3-day cultures). Both the cell bodies and the cell processes are stained. After 2-3 weeks, immunostainings of the neurones are very distinctive and bright; the non-neuronal cultured cells do not exhibit any labelling. These observations indicate the early acquisition of NIF and "It binding site expression by neuronal precursor cells (late gastrula stage). neural induction; neurofilament; tetanus toxin suppressed; binding site; neurone Introduction Morphogenetic movements during gastrulation lead to inductive neural interactions between the blastoporal lip (chordamesoderm, the neural in- ducer) and the presumptive ectoderm (the neural target tissue). Various studies in amphibians have shown that after in vivo or in vitro association with chordamesoderm, isolated presumptive ecto- ,4bbrevtali,m.~: FITC. fluorescein isothiocyanate: IF. inter- mediate filament: NF. neural fold: NIF. neurofilaments: NP. neural plate: Tt. tetanus toxin. dermal cells are able to differentiate into pheno- typical neurones in culture (Holtfreter, 1933: Jones and EIsdale, 1963: Gualandris and Duprat, 1981). However, the molecular mechanisms generating neuronal differentiation in terms of specific molec- ular marker expression have been little studied in amphibians (Vulliamy and Messenger, 1981: Duprat et al., 1984) and not a great deal is known about the first molecular events that occur after neural induction (cf. Spitzer, 1984: Warner. 1984: Duprat et al., 1985a, b). The main purpose of this study was to examine the time course of the appearance of two neuronal 0045-6039/86/$03.50 " 1986 ElsevierScientificPublishers Ireland, Ltd.