The appearance and localization of N-CAM during neural induction were studied in Pleurodeles waltl embryos and compared with recent contradictory results reported in Xenopus laevis.

A monoclonal antibody raised against mouse N-CAM was used. In the nervous system of Pleurodeles, it recognized two glycoproteins of 180 and 140 xlO3 which are the Pleurodeles equivalent of N-CAM-180 and -140.

Using this probe for immunohistochemistry and immunocytochemistry, we showed that N-CAM was already expressed in presumptive ectoderm at the early gastrula stage. In late gastrula embryos, a slight increase in staining was observed in the neurectoderm, whereas the labelling persisted in the noninduced ectoderm.

When induced ectodermal cells were isolated at the late gastrula stage and cultured in vitro up to 14 days, a faint polarized labelling of cells was observed initially. During differentiation, the staining increased and became progressively restricted to differentiating neurons.

Cell adhesion molecules or CAMs are cell surface glycoproteins which mediate cell-cell contact formation and play an essential role in the development and morphogenesis of multicellular organisms (Edelman, 1984). Several classes of cell adhesion molecules have now been identified and characterized (for reviews cf: Edelman, 1986; Takeichi, 1988). Among them, the neural cell adhesion molecule (N-CAM) has been defined as a primary CAM because of its early expression in embryonic development. Three size classes of N-CAM polypeptides have been identified with apparent relative molecular masses of 180, 140, and 120 x103. They differ mainly by the size of their cell-associated and cytoplasmic domains and are the results of alternative RNA splicing (Cunningham et al. 1987; Barbas et al. 1988).

As already shown in avian and mouse embryos, N-CAM expression changes during embryonic development (Thiery et al. 1982; Edelman et al. 1983; Klein et al. 1988). In the chick embryo, N-CAM was found to be first expressed at the blastoderm stage and then to become progressively restricted to the neuroepithelium during further development.

In amphibians, contradictory observations were recently reported with regard to the initial expression of N-CAM in Xenopus laevis embryos. Jacobson & Rutis-hauser (1986) and Balak et al. (1987), using an antibody against frog N-CAM, did not detect N-CAM prior to the neurula stage. In in vitro experiments involving combinations of the blastoporal lip (neural inducing tissue) with the competent presumptive ectoderm (neural target tissue), these authors showed that the association of both tissues was a prerequisite for N-CAM expression. Based on these results and on their in vivo localization studies, they concluded that N-CAM was expressed only after neural induction.

Kintner & Melton (1987) using a cDNA probe for N-CAM of Xenopus laevis, also showed that N-CAM gene expression was an early response to neural induction. By in situ hybridization on neurula-stage embryos, N-CAM mRNA was found exclusively in the neural plate, despite the fact that a low level N-CAM mRNA, probably of maternal origin, was detected before gastrulation by RNAse protection assays.

In striking contrast to these results, Levi et al. (1987), using different antibodies raised against Xenopus N-CAM, detected immunoreactive N-CAM prior to gastrulation. During gastrulation, its level was found to increase in the presumptive neuroepithelium, while it continued to be expressed weakly in the non-neural epidermis.

In the present study, we used a monoclonal antimouse N-CAM antibody that recognizes N-CAM-180 and -140 and that cross-reacts with N-CAM from many species to re-evaluate the expression of this adhesion molecule in the urodele amphibian Pleurodeles waltl during neural induction.

Embryos, obtained from natural matings of Pleurodeles waltl, were staged according to Gallien & Durocher (1957).

Cell cultures

Jellies were manually removed with fine forceps in Holtfreter’s solution. Competent presumptive ectoderm and the blastoporal lip were microsurgically excised from gastrulae in Holtfreter’s solution (Tris 5mm, pH8·5 including penicillin (100 i.u. ml-1) and streptomycin (100 μg ml-1)).

Using the classical sandwich method (Holtfreter, 1933), neural induction was provoked in vitro (Duprat et al. 1982). The induced ectoderm was dissociated into single cells in Ca2+-, Mg2+-free Barth’s medium pH 8·7 containing 0·5 HIm-EDTA. Isolated cells were then cultured for up to 14 days in Barth’s medium (Barth & Barth, 1959) supplemented with 0·1% bovine serum albumin (Sigma), on glass coverslips coated with collagen, in Nunc Petri dishes.

Neurectodermal cells induced in vivo were obtained from early neurula stage (stage 13) and cultured, together with the underlying chordamesodermal cells, as previously described (Duprat et al. 1985).

Cultures of presumptive ectodermal cells were used as controls. In these experiments, presumptive ectoderm was not previously associated with the blastoporal lip before dissociation and culture. This tissue differentiated into epidermal cell types.

Antibodies

The rat monoclonal antibody directed against mouse N-CAM (P61) has already been described (Gennarini et al. 1984a). Briefly, P61 is a rat IgG 2a that reacts with a domain located near or at the cytoplasmic side of the plasma membrane (Gennarini et al. 1984b) (Fig. 1) and recognizes selectively N-CAM-180 and -140 but not N-CAM-120.

Fig. 1.

Schematic representation of the different molecular forms of N-CAM. ▪ Localization of the epitope recognized by P61. N-CAM-180 and -140 are transmembrane molecules. N-CAM-120 is linked to the membrane by a phosphatidyl inositol (—○).

Fig. 1.

Schematic representation of the different molecular forms of N-CAM. ▪ Localization of the epitope recognized by P61. N-CAM-180 and -140 are transmembrane molecules. N-CAM-120 is linked to the membrane by a phosphatidyl inositol (—○).

Fig. 2.

Transverse section in the cephalic area of Pleurodeles waltl (stage 33), stained with P61 antibody. (A) N-CAM is expressed in the retinal cells (RC). This molecule is located at the cell plasma membrane. No staining is observed in the retinal pigmented epithelium (RPE) nor in the lens (L). (B) Corresponding phase-contrast micrograph. (C) Retinal cells, in the control section incubated with the non-specific myeloma supernatant H28.123, are negative. (D) Corresponding phase-contrast micrograph. Bar, 20 μm.

Fig. 2.

Transverse section in the cephalic area of Pleurodeles waltl (stage 33), stained with P61 antibody. (A) N-CAM is expressed in the retinal cells (RC). This molecule is located at the cell plasma membrane. No staining is observed in the retinal pigmented epithelium (RPE) nor in the lens (L). (B) Corresponding phase-contrast micrograph. (C) Retinal cells, in the control section incubated with the non-specific myeloma supernatant H28.123, are negative. (D) Corresponding phase-contrast micrograph. Bar, 20 μm.

The rat monoclonal antibody, H28.123 (Him et al. 1981; Gennarini et al. 1984b), which is known to bind the three forms of N-CAM in mouse but none of these molecules in Pleurodeles (Fig. 3, lane 6), was used as control in all cases.

Fig. 3.

Biochemical characterization of the antibody, P61, used in this study. Immunoblotting of adult Pleurodeles tissue extracts after fractionation by SDS-PAGE. Lane I: Adult liver extract (100μg proteins per well). Lane 2: Adult brain extract (100 μg proteins per well). Lane 3: Immunoprecipitation of adult brain extract. Lane 4: Control of immunoprecipitation omitting tissue extract. Lane 5: Control on adult brain extract omitting the primary antibody (P61). Lane 6: Control on adult brain extract using the non-specific myeloma supernatant-H28,123.

Fig. 3.

Biochemical characterization of the antibody, P61, used in this study. Immunoblotting of adult Pleurodeles tissue extracts after fractionation by SDS-PAGE. Lane I: Adult liver extract (100μg proteins per well). Lane 2: Adult brain extract (100 μg proteins per well). Lane 3: Immunoprecipitation of adult brain extract. Lane 4: Control of immunoprecipitation omitting tissue extract. Lane 5: Control on adult brain extract omitting the primary antibody (P61). Lane 6: Control on adult brain extract using the non-specific myeloma supernatant-H28,123.

A second control could be performed using P61 antibody exhausted with purified antigen. N-CAM from adult mouse brain (100 μgm11) was purified as described (Gennarini et al. 1984a) and incubated with P61 antibody at about 50 μgm11 overnight at 4°C. Formed complex were pelleted by centrifugation and the supernatant used as control.

Fluorescein isothiocyanate-conjugated rabbit anti-rat-IgG (FITC/RARa) was purchased from Nordic (Tilburg, the Netherlands).

Neuronal population in culture was characterized using double-staining experiments performed with P61 and detection of tetanus-toxin-binding site (Duprat et al. 1986).

Immunohistochemistry

This study was performed on embryos at stage 8a (early gastrula stage), stage 14 (early neurula stage) and stage 33 (swimming larvae). Embryos were fixed in 3·5% formaldehyde in Barth’s medium pH 7·4 for 12 h at 4 °C. After thorough washing in Barth’s medium, they were successively impregnated for Ih each in 5%, 10%, 15% and 20% sucrose, for 12h in 25 % sucrose and for at least 3 days in 30 % sucrose in Barth’s medium at 4°C. Embryos were embedded in OCT compound (Tissue Tek/Miles Scientific), and immediately immersed in isopentane previously cooled to —80°C by liquid nitrogen. Sections of 14μm (frigocut 2800-Reichertβung) were mounted on glass slides coated with gelatin. Sections were preincubated in PBS containing 1% dry skim-milk (PBS -I-M) for 30 min, then reacted overnight at 4 °C with P61 in the form of hybridoma supernatant (diluted 1/10 in PBS + M) and revealed by FITC-labelled secondary antibody (diluted 1/50 in PBS + M) for 30 min. Each step was followed by extensive washings in PBS + M. Sections were mounted in Mowiol 4-88 and observed with an epifluor-escence Leitz-Dialux microscope (Filter I2 BP 450-490, LP 515).

Immunocytochemistry

For N-CAM visualization, cell cultures were fixed in 3·5% formaldehyde in Barth’s medium for 30 min at 20 °C, then washed in Barth’s medium containing 1 % dry skim-milk (BM + M) and incubated with P61 for 30min at 20°C. After washing, bound antibody was revealed by FTTC-RARa for 30min. Slides were mounted in Mowiol.

For double-staining experiments, cell cultures were first incubated with tetanus toxin (10 μg ml-1) for 10 min, revealed successively with rabbit anti-tetanus toxin antibody and with goat anti-rabbit antiserum conjugated with tetramethyl-rhodamine isothiocyanate (GAR-TRITC) as described (Duprat et al. 1986), then fixed and processed for P61 staining.

Immunoblotting

Pleurodeles adult brain or liver were dissected and homogenized on ice in 100mm-NaCI, 50mm-Tris-HCI pH7·4, 0·1 mm-phenylmethyl sulfonyl fluoride (PMSF, Sigma), and 0·5% Nonidet P40 (Sigma). Homogenates were maintained for 10 min on ice, then centrifuged at 13000g for 10 min at 4 °C.

The supernatants were either directly boiled in SDS sample buffer or N-CAM was first immunoprecipitated by successive incubations with P61 antibody (1 h at 20°C) and A-Sepharose-complexed rabbit anti-rat 1g (16 h at 4 °C). After several washings in NaCl 100mm, Tris-HCl 50 mm pH 7·4, EDTA 5 mm and 0·lmm-PMSF, the Sepharose beads were boiled in SDS sample buffer.

After fractionation by SDS-PAGE (Laemmli, 1970), the proteins were electroblotted to nitrocellulose paper (Towbin et al. 1979).

Nitrocellulose sheets were saturated with 5 % dry skim-milk in PBS pH 7 4 (PBS + M) for 4h at 20°C. Incubation with P61 (1/10 in PBS + M) was performed overnight at 4°C. Bound antibody was then visualized with peroxidasc-conjugated RARa IgG (1/1000 in PBS + M) for 4h at 20°C. Peroxidase-conjugated antibodies were revealed using 3-3’di-aminobenzidine (DAB, Sigma) and H2O2.

Relative molecular mass markers (Biorad) were of 200,116, 97, 66 and 42X103, respectively.

The antibody used was directed against mouse N-CAM. In order to test its reactivity on Pleurodeles, we used two different approaches: an immunohistochemical study and an immunoblotting study on neuronal and non-neuronal tissues.

In frozen sections of larvae (stage 33), a strong reactivity was detected in the cephalic area both in the brain and on retinal cells, where the fluorescent pattern indicated plasma membrane staining (Fig. 2, A,B). No reactivity with P61 was observed either in non-neuronal tissues in these sections (lens, retinal pigmented epithelium and epiderm) or in adult liver sections (data not shown). A control using the nonspecific myeloma supernatant (H28.123) gave no signal (Fig. 2, C,D).

In immunoblots done with brain extracts of adult Pleurodeles, P61 mainly stained a 180x103Mr (180K) band strongly (Fig. 3, lane 2). After enrichment of the immunoreactive components by immunoprecipitation, (Fig. 3, lane 3) the 140K component became clearly visible. No P61-reactive proteins were seen in liver extracts of adult Pleurodeles (Fig. 3, lane 1). All the control experiments performed were negative (Fig. 3, lanes 4, 5 and 6).

Expression of N-CAM during gastrulation

Indirect immunofluorescence was used to study the localization of N-CAM in Pleurodeles waltl embryos during neural induction stages.

At the first stage examined, i.e the early gastrula (stage 8a) before neural induction, a low level of fluorescence was detected in the presumptive ectoderm, at the level of the plasma membrane (Fig. 4 A,B). Staining of endodermal and chordamesoderma! cells was also observed. This fluorescence pattern was not observed with the nonspecific myeloma supernatant, H28.123. A second control using P61 antibody exhausted with purified mouse N-CAM gave no signal (Fig. 4C). The very low autofluorescence of yolk platelets did not interfere with the specific staining.

Fig. 4.

Early gastrula (stage 8a) section stained with P61 antibody. (A) A weak fluorescence is detected in the competent presumptive ectoderm, where it is mainly located at at the cell membrane (arrowheads). (B) Corresponding phase-contrast micrograph. Blastocoele (BL). (C) Control staining using P61 antibody exhausted with purified N-CAM shows that P61 labelling is clearly above background. Bar, 20 μm.

Fig. 4.

Early gastrula (stage 8a) section stained with P61 antibody. (A) A weak fluorescence is detected in the competent presumptive ectoderm, where it is mainly located at at the cell membrane (arrowheads). (B) Corresponding phase-contrast micrograph. Blastocoele (BL). (C) Control staining using P61 antibody exhausted with purified N-CAM shows that P61 labelling is clearly above background. Bar, 20 μm.

At the late gastrula stage, a slight increase in P61 staining was observed in the neurectoderm. Cells of the inner surface became more brightly fluorescent than the cells of the underlying chordamesoderm (Fig. 5, A,B). Once again the immunoreactivity seemed to be localized at the plasma membrane. No difference in labelling was observed between the two areas of the neurectoderm: the neural plate and the neural fold. N-CAM continued to be expressed in the lateral and ventral ectoderm, although this diffuse non-neural staining disappeared later in development (stage 33), the labelling was then restricted to neuronal cells. Controls using the nonspecific myeloma supernatant H28.123 gave no signal as shown in Fig. 5 (C,D).

Fig. 5.

Early neurula (stage 14) section stained with P61 monoclonal antibody. (A) N-CAM is clearly expressed in the neurectoderm. A sharp limit can be observed between the neurectoderm and the underlying chordamesoderm (arrowheads). (B) Corresponding phase-contrast micrograph. Neurectoderm (NE), chordatnesoderm (CM). (C) The irrelevant antibody H28.123 gave no signal on neurectodermal cells. (D) Corresponding phase-contrast micrograph. Bar, 20 μm.

Fig. 5.

Early neurula (stage 14) section stained with P61 monoclonal antibody. (A) N-CAM is clearly expressed in the neurectoderm. A sharp limit can be observed between the neurectoderm and the underlying chordamesoderm (arrowheads). (B) Corresponding phase-contrast micrograph. Neurectoderm (NE), chordatnesoderm (CM). (C) The irrelevant antibody H28.123 gave no signal on neurectodermal cells. (D) Corresponding phase-contrast micrograph. Bar, 20 μm.

In vitro expression of N-CAM

Presumptive ectoderm and neurectoderm obtained either after in vivo or in vitro induction, were cultured after dissociation into single cells up to 1,4,7,12 and 14 days. They were then treated for immunocytochemistry with the P61 antibody to study the time-course of appearance of positive immunoreactivity.

Culture of presumptive ectodermal cells

Freshly dissociated cells of the presumptive ectoderm (stage 8a) displayed a low level of staining, polarized to one side of the cell which may correspond to the lobopod (Fig. 6 A,B), a specialized structure which plays a role in cell attachment (Holtfreter, 1947).

Fig. 6.

Expression of N-CAM on presumptive ectodermal cells cultured in vitro.

(A,B) Freshly dissociated cell. (A) Labelling is polarized to one side of the cell, possibly the lobopod (arrowhead). (B) Corresponding phase-contrast micrograph. Bar, 10 μm. The cells are rich in yolk platelets. (C,D) 1-day-old culture. (C) Some pseudopodia on one small aggregate are faintly stained (arrowheads), whereas cells in the aggregate lack immunoreactivity for P61. (D) Corresponding phase-contrast micrograph. Bar, 20 μm. (E,F) 12-day-old culture. (E) Epidermal sheet presenting no reactivity to P61. (F) Corresponding phase-contrast micrograph. Cells have now digested most of their vitellus. Bar, 20 μm.

Fig. 6.

Expression of N-CAM on presumptive ectodermal cells cultured in vitro.

(A,B) Freshly dissociated cell. (A) Labelling is polarized to one side of the cell, possibly the lobopod (arrowhead). (B) Corresponding phase-contrast micrograph. Bar, 10 μm. The cells are rich in yolk platelets. (C,D) 1-day-old culture. (C) Some pseudopodia on one small aggregate are faintly stained (arrowheads), whereas cells in the aggregate lack immunoreactivity for P61. (D) Corresponding phase-contrast micrograph. Bar, 20 μm. (E,F) 12-day-old culture. (E) Epidermal sheet presenting no reactivity to P61. (F) Corresponding phase-contrast micrograph. Cells have now digested most of their vitellus. Bar, 20 μm.

In 1-day-old cultures, cells were attached to the substrate and most of them participated in the formation of large epithelial structures which lacked immunoreactivity for P61. Only some pseudopodia on small aggregates were faintly stained (Fig. 6C,D). The fluorescence observed on freshly dissociated cells now seemed to be concentrated on pseudopodia.

In 4-day-old cultures, the by now well differentiated epidermal epithelium remained totally negative with P61. Identical negative patterns were observed in 7- and in 12-day-old cultures (Fig. 6E,F). In all cases studied, controls using the irrelevant antibody H28.123 were negative.

Culture of neurectodermal cells

Results were similar irrespective of whether neural induction occurred in vivo or in vitro.

Immediately after induction, dissociated cells showed a staining pattern identical to that of cells originating from a presumptive ectoderm, i.e. a labelling indicative of the lobopod (Fig. 7 A,B).

Fig. 7.

Expression of N-CAM on neurectodermal cells cultured in vitro. (A,B) Freshly dissociated cells. (A) Dissociated cells of freshly induced ectodermal cells present an immunoreactivity to P61 indicative of the lobopod (arrowhead). (B) Corresponding phase-contrast micrograph. Bar, 10 μm. (C,D) 4-day-old culture. (C) No immunoreactivity to P61 is detected on melanocyte (arrow) and mesenchymal cells. (D) Corresponding phase-contrast micrograph. Bar, 20 μm. Melanocyte (arrow). (E,F/G,H) 7-day-old culture. (E) Small neuronal aggregate presenting a strong reactivity to P61. Staining is mostly situated at the cell membrane (arrowheads); neuritic process are poorly stained (arrow). (F) Corresponding phase-contrast micrograph. Bar, 20 μm. (G) Neurons are negative with the non-specific myeloma supernatant H28.123. (H) Corresponding phase-contrast micrograph. Bar, 20 μm.

Fig. 7.

Expression of N-CAM on neurectodermal cells cultured in vitro. (A,B) Freshly dissociated cells. (A) Dissociated cells of freshly induced ectodermal cells present an immunoreactivity to P61 indicative of the lobopod (arrowhead). (B) Corresponding phase-contrast micrograph. Bar, 10 μm. (C,D) 4-day-old culture. (C) No immunoreactivity to P61 is detected on melanocyte (arrow) and mesenchymal cells. (D) Corresponding phase-contrast micrograph. Bar, 20 μm. Melanocyte (arrow). (E,F/G,H) 7-day-old culture. (E) Small neuronal aggregate presenting a strong reactivity to P61. Staining is mostly situated at the cell membrane (arrowheads); neuritic process are poorly stained (arrow). (F) Corresponding phase-contrast micrograph. Bar, 20 μm. (G) Neurons are negative with the non-specific myeloma supernatant H28.123. (H) Corresponding phase-contrast micrograph. Bar, 20 μm.

After 1 day in culture, the typical ‘induced behaviour’ of cells was observed (Gualandris & Duprat, 1981). Briefly, committed cells spread and remained isolated on the substrate or formed small loose clusters. Labelling was observed at the periphery of these small aggregates.

In 4-day-old cultures, in which morphological differentiation was occurring, several different cell types were recognizable. These included melanocytes, mes-enchymal cells, fibroblast-like cells, muscle cells and neurons. None of the non-neural cell types were immunoreactive with P61 (Fig. 7C,D). It was only after 7 days of culture that an intense specific labelling was observed on neuronal cells. The plasma membranes of nerve cell bodies were brightly fluorescent, whereas neurite outgrowths were poorly stained (Fig. 7E,F). The fluorescence persisted on neuronal aggregates as differentiation proceeded. Labelling was detected on neuronal cells that had aggregated, either into large or small clusters originating from the neural plate and the neural fold, respectively (Duprat et al. 1985). Neuronal cells processed with H28.123 antibody were negative (Fig. 7G,H).

Double-staining experiments performed with P61 and tetanus toxin confirmed this specific labelling observed on neuronal cells (Fig. 8 A-C).

Fig. 8.

Double-staining experiment on neurectodermal cells cultured in vitro for 14 days. (A) Neuronal aggregate stained with P61 displayed a labelling characteristic of cell body plasma membrane (arrowheads). (B) Same neuronal aggregate stained with tetanus toxin. The labelling corresponds to the pattern observed with P61 (arrowheads). (C) Corresponding phase-contrast micrograph. Bar, 20 μm.

Fig. 8.

Double-staining experiment on neurectodermal cells cultured in vitro for 14 days. (A) Neuronal aggregate stained with P61 displayed a labelling characteristic of cell body plasma membrane (arrowheads). (B) Same neuronal aggregate stained with tetanus toxin. The labelling corresponds to the pattern observed with P61 (arrowheads). (C) Corresponding phase-contrast micrograph. Bar, 20 μm.

The distribution of N-CAM was examined in Pleurodeles waltl in relation to neural induction, using a monoclonal antibody raised against mouse N-CAM, P61. By immunohistochemistry it was shown that this antibody decorated the nervous system of adult Pleurodeles. In immunoblots of adult brain extracts, P61 bound two proteins with apparent relative molecular masses of 180 and 140X103, which are the Pleurodeles equivalents of N-CAM-180 and -140.

In vivo, the expression of N-CAM was examined prior (early gastrula stage) and subsequent (neurula stage) to neural induction. Immunohistochemically, N-CAM could already be observed in the whole embryo, more particularly in the presumptive ectoderm, at the onset of gastrulation Its expression was slightly enhanced in the neuroepithelium after neural induction At this stage, N-CAM was still expressed in the lateral and ventral ectoderm.

Studies on the in vitro expression of N-CAM showed that dissociated cells of the presumptive ectoderm, induced (by contact with inducing tissue) or noninduced (not associated with the inducing tissue), exhibited the same low level of staining that characterized these cells in vivo. As cultures proceeded, ectodermal cells progressively lost their labelling and become totally negative from 4 days onward. In contrast, in neurectodermal cell cultures, a clearly detectable staining by P61 was observed at 7 days. This immunoreactivity was restricted to the neuronal cell population as shown by doublestaining experiments; non-neuronal cells were negative. This specific neuronal staining was concomitant with the acquisition of various specific phenotypic markers such as tetanus-toxin-binding sites and the polypeptides of the neurofilaments (Duprat et al. 1986).

In Xenopus laevis, differences in the patterns of N-CAM expression have been reported. Jacobson & Rutishauser (1986) and Balak et al. (1987), using a rabbit polyclonal antibody against frog N-CAM that apparently recognizes essentially N-CAM-180, first detected N-CAM at the neurula stage where the staining was confined to the cells of the neurectoderm. On the other hand, Levi et al. (1987), with a rabbit polyclonal antibody that recognizes all three N-CAM proteins, have shown that N-CAM is detectable from the 2-cell stage onwards. After gastrulation, an increase in N-CAM staining was observed in the neuroepi-thelium. According to these authors, the component detected prior to gastrulation is the 140K form.

Similarly, using a probe potentially recognizing transcripts for all N-CAM isoforms, Kintner & Melton (1987) were able to detect N-CAM mRNA in the fertilized egg, by RNAse protection assays, suggesting a maternal origin of early expressed N-CAM mRNA. However, an increase in N-CAM mRNA was only detectable after gastrulation. In situ hybridization localized this last increased expression to the neurectoderm, in agreement with the results of Jacobson & Rutishauser (1986) and of Balak et al. (1987). Taken together, the different data previously reported for Xenopus laevis can be explained by assuming that N-CAM-140 is expressed first in development, while the expression of the 180K form requires neural induction.

Our results, obtained in a different species, agree with those described by Levi et al. (1987) on the early expression of N-CAM. The antibody we used detected both N-CAM-180 and N-CAM-140, the ability to identify the latter isoform enabling us to visualize this N-CAM form prior to gastrulation and after neural induction in the non-neural ectoderm.

In contrast to the observations reported by Levi et al. (1987), who detected a 160K protein in Xenopus liver, we did not detect immunoreactivity to P61 in the liver of Pleurodeles. Although we cannot exclude the possible presence of an N-CAM form lacking the P61 epitope, on this point urodele amphibian seems to be nearer to chicken and mouse than to anurans.

With regard to N-CAM expression, our results from in vivo and in vitro studies are similar to those reported in the avian embryo (Thiery et al. 1982; Edelman et al. 1983; Murray et al. 1986) and in the mouse (Pollerberg et al. 1985). They point out that the N-CAM with a small cytoplasmic domain (N-CAM-140) is the first to be expressed in the embryo. During development of the nervous system, N-CAM-140 continues to be expressed at a low level; N-CAM-180, with a large cytoplasmic domain, seems associated exclusively with differentiating neurons.

The authors would like to thanks Dr J. Smith for reviewing the English manuscript, Dr J. P. Thiery for stimulating discussions and Mrs C. Daguzan for photographic assistance. This work was supported by grants from the CNRS, the MRES and the EEC.

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