Amongst the many cell types that differentiate from migratory neural crest cells are the Schwann cells of the peripheral nervous system. While it has been demonstrated that Schwann cells will not fully differentiate unless in contact with neurons, the factors that cause neural crest cells to enter the differentiative pathway that leads to Schwann cells are unknown. In a previous paper (Development 105: 251, 1989), we have demonstrated that a proportion of morphologically undifferentiated neural crest cells express the Schwann cell markers 217c and NGF receptor, and later, as they acquire the bipolar morphology typical of Schwann cells in culture, express S-100 and laminin. In the present study, we have grown axons from embryonic retina on neural crest cultures to see whether this has an effect on the differentiation of neural crest cells into Schwann cells. After 4 to 6 days of co-culture, many more cells had acquired bipolar morphology and S-100 staining than in controls with no retinal explant, and most of these cells were within 200 μm of an axon, though not necessarily in contact with axons. However, the number of cells expressing the earliest Schwann cell markers 217c and NGF receptor was not affected by the presence of axons. We conclude that axons produce a factor, which is probably diffusible, and which makes immature Schwann cells differentiate. The factor does not, however, influence the entry of neural crest cells into the earliest stages of the Schwann cell differentiative pathway.

During vertebrate development, neural crest (NC) cells migrate from the dorsal aspect of the neural tube into many regions of the embryo where they differentiate into a wide variety of cell types including most of the Schwann cells and neurons of the peripheral nervous system (see review by Le Douarin, 1982). Avian and mammalian neural crest cells will differentiate in vitro, and the type of cell into which they differentiate can be controlled to some extent by the tissue culture substrate, by soluble factors in the medium and by combination with other cell types. Examples are found in the control of skeletogenic (Bee and Thorogood, 1980; Smith and Thorogood, 1983), neurogenic (Le Douarin, 1982; Smith-Thomas et al. 1986), melanogenic (Derby and Newgreen, 1982) and odontoblastic (Lumsden, 1984) differentiation.

Schwann cells will only differentiate fully when in contact with axons or neurons (Bunge et al. 1982, 1983; Holton and Weston 1982a,b). Until recently it was not known at what stage NC cells start to differentiate into Schwann cells and whether they need to be in contact with neurons at this time. We have recently demonstrated that a proportion of cultured rat premigratory neural crest cells express two markers that are also found on mature Schwann cells, NGF receptor and 217c (Smith-Thomas and Fawcett, 1989), and that premigratory and migrating neural crest cells in vivo express these same markers (Smith-Thomas and Fawcett, unpublished); this suggests that the first stages of Schwann cell differentiation occur before the cells have contacted axons or neurons. These markers, 217c and NGF receptor are first seen on morphologically undifferentiated cells, which will later acquire the bipolar morphology and S-100 staining typical of more differentiated Schwann cells (Smith-Thomas and Fawcett, 1989; Holton and Weston, 1982a,b). 217c and NGF receptor must mark, therefore, the earliest stages of Schwann cell differentiation. Neither of these two markers is restricted entirely to Schwann cells; monoclonal 217c binds to a cell surface antigen found on many rat nervous system tumour cell lines (Peng et al. 1982; Fields and Dammerman, 1985), on cultured rat Schwann cells (Brockes et al. 1977, 1979) and on cultured dorsal root ganglion neurons (Smith-Thomas and Fawcett, 1989). Nerve growth factor receptor is present on Schwann cells deprived of axonal contact in vivo and in vitro, on many neuronal cell surfaces, and is recognised by monoclonal antibody 192-IgG (Zimmerman and Sutter, 1983; Taneuchi et al. 1986; DiStefano and Johnson, 1988). Since both Schwann cells and dorsal root ganglion (DRG) neurons stain with 217c and NGF receptor, and both are neural crest derived, it is possible that both cell types share the same precursor cell. However, the close association between Schwann cells and neurons observed in our rat neural crest cultures also suggested to us that neurons might be involved in early Schwann cell differentiation.

The aim of the present experiments was to establish whether neurons and their processes can promote the differentiation of NC cells into Schwann cells. In order to do this, we cultured premigratory rat neural crest cells and then placed expiants of embryonic retina on top of them, the axons of which grew extensively on the NC cells. We then quantified the effects of the retinal neurons and their processes on neural crest cell differentiation into Schwann cells, using the antibodies that have been shown to mark stages in Schwann cell differentiation.

Neural crest cultured alone

The procedure for preparation of rat trunk neural crest cultures has been described in detail elsewhere (Smith-Thomas and Fawcett, 1989). Briefly, neural tubes were removed from 18 to 24 somite rat embryos and isolated from surrounding tissues by enzyme digestion and mechanical dissociation. Neural tubes were then washed twice with medium and plated onto glass coverslips coated with 0.01 % polylysine (Sigma) and 25 μgml−1 fibronectin (Sigma) and pre-incubated for lh in medium. The medium comprised Eagles alpha MEM (Gibco)+10% FCS (Flow)+5% of whole chick embryo extract (Flow)+penicillin and streptomycin. Two neural tubes were plated onto each coverslip and left to attach for 1 to 2 h. After the neural tubes had attached, 1.5 ml of medium was added to each 35 mm culture dish. On the second or third day of culture, at which time a sizable neural crest outgrowth was present, the neural tube was removed from the culture using a tungsten needle and the cultures were fed with either 1- to 2-day or 6- to 14-day neural tube conditioned medium. After 5 to 7 days of culture, the medium was removed and replaced with DMEM +10% foetal calf serum (FCS) and 0.7% methylcellulose (i.e. medium identical to that used in NC+retina cultures).

The neural crest cell morphology was examined every day using an inverted phase-contrast microscope. After a total culture period of 10 or 13 days, the cultures were fixed for immunocytochemistry.

El 6 retina cultured alone

Retinas were dissected from E16 rat eyes (plug day=l) and cut into 3 or 4 pieces using a pair of iridectomy scissors. These were cultured on glass coverslips coated with polylysine (0.01%) and laminin (10/zgml-1) in medium containing methylcellulose. After a total culture period of 4 to 7 days, the cultures were fixed for immunocytochemistry.

NC+E16 retina co-cultures

Neural crest was cultured for 5 to 7 days. The medium was then replaced with medium containing methylcellulose and a single piece of E16 retina was then placed on top of the NC (a region of homogeneous mesenchymal cells at a distance from any CNS tissue). The tissues were co-cultured for 6 to 7 days and the morphology of the neural crest cells was examined every day using an inverted phase-contrast microscope. After a total culture period of 10 to 13 days, the cultures were fixed for immunocytochemistry.

Removal of neurons from neural crest cultures using Tetanus toxin and A2B5 complement-mediated lysis

Neural crest cells were cultured for 5 or 6 days. Cultures were washed in warm sterile Hanks and then incubated in tetanus toxin 20μgml−1 (Calbiochem) and A2B5 monoclonal supernatant 1:5 at 37°C for 30min, washed, and then incubated in a mixture of rabbit antitetanus toxin 1:50 (gift from Dr R. Thomson, Wellcome labs) and complement for 40 min at 37°C.

Staining procedures

S-100+RT97

Cultures were washed twice with PBS/1% BSA and then fixed for 15min in 4% paraformaldehyde pH7.3. Cultures were then washed three times with PBS/1 % BSA, blocked with PBS/0.2% Triton/5% goat serum for 30 min and then incubated in a mixture of antibody to S-100 (1:200) (Dako)+RT97 (1:200) (gift from John Wood) in PBS/Triton/1 % goat serum for 1h at room temperature. The cultures were then washed twice with PBS/1% BSA and then incubated in biotinylated goat anti-mouse (BRL) for 30min. The cultures were then washed twice in PBS and then incubated in a mixture of FITC-conjugated goat anti-rabbit (l:50)(Tago) and RITC-streptavidin (1:100) (Serotec) for 30min. The cultures were then washed three times in PBS and mounted in glycerol/PBS (1:1).

m217c and 192-IgG anti-NGF receptor

Cultures were washed twice with PBS/1% BSA and then fixed for 15 min in 4% paraformaldehyde. They were then washed three times with PBS/1% BSA, blocked with PBS/5 % goat serum for 30 min and then incubated in m217c supernatant diluted 1:80 or 192-IgG at 2μgml−1 in PBS/1% goat serum for 1h. Cultures were then washed three times in PBS/1% BSA followed by biotinylated goat anti-mouse (1:50) (Gibco BRL) for 30 min at RT. The cultures were then washed twice in PBS and incubated with FITC-streptavidin (1:100) (Serotec), washed three times in PBS and mounted in glycerol/PBS (1:1). The m217c supernatant was a gift from Dr J. de Vellis, and 192-IgG was a gift from Dr E. Johnson.

Quantification of S-100 and m217c stained and bipolar cells in neural crest cultured in presence or absence of E16 retina and estimation of significance of results

(i) Neural crest cultured alone: controls

Neural crest was cultured for 6 or 7 days on coverslips that had two scratch marks on the edge at 90° to each other so that, using a calibrated graticule, a particular region of the culture could be localized early in the culture period and then referred back to at the end of the culture period. The region of the culture that was localized was a large area of homogeneous mesenchymal cells that was free of any CNS tissue (i.e. an area similar to where the piece of retina would be put at this time) and was referred to as point A. After a total culture period of 10 to 13 days, the culture was fixed and stained for m217c, S-100 and RT97. Hie point A was then referred back to. Using a calibrated square graticule, the number of m217c-stained, bipolar, S-100-containing and total cells visible in a 250 μm square were counted at 500 μm intervals. Cells were counted from point A right to left to the edge of the NC culture (point B) and then from A left to right to the other edge of the culture (point C). The % number of each cell type at each distance (expressed as a % of the total number of cells visible in the 250 μm square graticule) was then estimated. The mean number of each cell type as a % of the total cells within 1000/mi intervals and the S.D. was then estimated by adding up the % number of each cell type obtained for all the cultures within each 1000pm interval and then estimating the mean and S.D. for these values (pooling the results from A to B and A to C).

(ii) Neural crest and retina co-cultures

After fixation and staining, a central point on the edge of the retinal explant was selected (point A) and the number of each cell type was counted as in the neural crest cultured alone. The mean and S.D. for each cell type as a % of the total cells within 1000 μm intervals was then determined as described above for neural crest cultured alone.

Statistics

The experiments described in this paper were designed to test the hypothesis that retinal neurons would cause neural crest cells to acquire a bipolar morphology and express S-100 protein. Thus, a onetailed t-test was performed on the data to assess the significance of the results for these cellular characteristics. The level of significance between the experimental and control cell counts was estimated for each 1000 μm interval from point A. There was no prior hypothesis as to whether the retinal neurons would cause the number of m217c-staining cells to increase or decrease and thus a two-tailed t-test was performed on this data. In addition, we conducted a two-factor analysis of variance with the different culture types as a between-culture factor and distance from point A (see above) as a within culture factor.

Neural crest cultured alone

The morphology of rat neural crest cultures has been described previously (Smith-Thomas and Fawcett, 1989). Morphologically, the majority of cells are mesenchymal in morphology until day 10 of culture, by which time patches and occasionally nests of bipolar cells and a few cells of neuronal morphology are present, increasing numbers of cells differentiating with time. However, immunostaining with neuronal and Schwann cell markers reveals that cells have begun to differentiate earlier than this. A proportion of flat mesenchymal cells express NGF receptor and stain with m217c from the beginning of the culture; then at about 7 days some cells express the neuronal marker neurofilament (NF) and can be stained with tetanus toxin. At 12 days, there are nests of bipolar cells some of which begin to express S-100 protein, and these cells are usually close to or in contact with cells of neuronal type (Smith-Thomas and Fawcett, 1989). The neurons that differentiate in the culture do not stain with A4 antibody, which only stains neurons and glia of CNS origin (Cohen and Selvendran, 1981): there are a few A4-positive cells restricted to the precise region of the original neural tube explant.

We attempted to remove the neurons that differentiated in neural crest cultures, because there is some Schwann cell differentiation around them. To do this we used tetanus toxin and anti-toxin and monoclonal A2B5 and complement. This killed all the cells of neuronal morphology, and treated cultures stained 1 day later with antibodies to NF or with tetanus toxin had very few immunoreactive cells in them. However, over time some cells expressing neuronal markers reappeared, some with a mesenchymal morphology, and some with a typical neuronal morphology. Some cultures were given a second tetanus toxin and complement treatment and neurons still differentiated in these cultures. These observations indicate that, although tetanus toxin and A2B5 complement mediated lysis kills neurons present in the cultures, neurons continue to differentiate from the neural crest cells.

16 retina cultured alone on laminin

We cultured retinal expiants by themselves in order to observe the migration of non-neuronal cells. The expiants attached and produced axons within 24 h and for the 9 days over which we observed expiants, there was prolific axonal outgrowth (Fig. 1A), and a small amount of non-neuronal cell migration, which extended for a distance of up to 2500 μm. The non-neuronal cells were flat, mesenchymal and did not stain with m217c or S-100.

Fig. 1.

(A) E16 day retina cultured on laminin for 6 days. Large numbers of axons have extended from the retinal expiant (r). Bar=100μm. (B) Neural crest cultured for 7 days and then co-cultured with E16 retina (r) for 6 days. Large numbers of bipolar cells (arrows) are associated with the axons. The round phase-bright cells are probably mitotic cells. Bar=200μm. (C) Neural crest cultured for 7 days and then co-cultured with E16 retina for 4 days. Culture fixed and stained with m217c. Large numbers of bipolar cells in the neural crest culture are stained with m217c. Bar=100μm.

Fig. 1.

(A) E16 day retina cultured on laminin for 6 days. Large numbers of axons have extended from the retinal expiant (r). Bar=100μm. (B) Neural crest cultured for 7 days and then co-cultured with E16 retina (r) for 6 days. Large numbers of bipolar cells (arrows) are associated with the axons. The round phase-bright cells are probably mitotic cells. Bar=200μm. (C) Neural crest cultured for 7 days and then co-cultured with E16 retina for 4 days. Culture fixed and stained with m217c. Large numbers of bipolar cells in the neural crest culture are stained with m217c. Bar=100μm.

NC co-cultured with E16 retina

In order to see whether neuronal tissue would increase the differentiation of NC cells into Schwann cells, we placed expiants of E16 retina on the flat mesenchymal NC cells that had migrated out from the neural tubes. Some cultures received retinal expiants and, in the others, which were our controls, we took the coordinates of a point on the culture similar to that where we would have put a retinal explant, and later used this point as the ‘centre’ (point A-see methods) from which to perform our control cell counts. The neural crest cultures were allowed to grow for 5 to 7 days before retina was added, at which time the NC cell outgrowth was entirely flat and mesenchymal in appearance. After 4 to 6 days of co-culture, the cultures were fixed and stained.

1 day after placing the retina on to the neural crest culture, neurites had grown out over the neural crest cells, but the cells remained flattened and mesenchymal in morphology. Axonal growth was profuse, and reached an average of 3500μm from the explant by 6 days. However, 2 or 3 days after addition of the retina, large numbers of neural crest cells adjacent to the retinal axons had acquired a bipolar morphology, which persisted for the rest of the culture period (Fig. IB). Axons and associated bipolar cells appeared to be lying on top of and beside flattened cells.

4 days after addition of the retina to the NC cultures, we counted the number of cells of bipolar morphology. There were significantly more bipolar cells at a distance of 0 to 1000μm from A, a central point on the edge of the retinal explant compared to a similar position in control cultures (see Table 1 and Fig. 2A). We also stained co-cultures for m217c 4 days after addition of the retina. As we reported previously, there were many 217c-stained cells throughout both experimental and control cultures, mostly of flat mesenchymal morphology. However, in the experimental group many of the m217c-stained cells near the retinal explant were bipolar in morphology, were usually lined up end to end in a sheet-like configuration, and looked very like cultured Schwann cells (Fig. 1C) (Brockes et al. 1977). However, although there were significantly more cells which were both bipolar and stained with m217c near the retinal explant by r-test, there was no significant difference in the overall number of m217c-stained cells in experimental and control cultures (see Table 2 and Fig. 2B). When a two-factor analysis of variance was conducted, the effect of the retina on bipolar morphology was highly significant compared to either neural crest cultured alone or in astrocyte-conditioned medium (see later) (main effect due to culture type, F=14.64, with 1,7 df, P<0.006; main effect due to distance from point A, F=10.35 with 4,28 df, P<0.001; culture typexdistance interaction, F=5.66 with 4,28df P<0.002. The effect of the retina on the number of neural crest cells that were m217c immunoreactive was not significant compared to either neural crest cultured alone or in astrocyte-conditioned medium (main effect due to culture type, F=0.21 with 1,7 df n.s.; culture typexdistance interaction, F=0.37 with 4,23df, n.s.). The percentage of m217c-stained cells was higher closest to point A in both culture types (main effect of distance F=4.74, with 4,23df, P<0.006).

Table 1.

Bipolar cells in neural crest cultured for 6 or 7 days and then in presence or absence of E16 retina for 4 days (total NC culture time=10 or 11 days)

Bipolar cells in neural crest cultured for 6 or 7 days and then in presence or absence of E16 retina for 4 days (total NC culture time=10 or 11 days)
Bipolar cells in neural crest cultured for 6 or 7 days and then in presence or absence of E16 retina for 4 days (total NC culture time=10 or 11 days)
Table 2.

m217c-positive cells in neural crest (NC) cultured for 6 or 7 days and then in presence or absence of E16 retina for 4 days (total NC culture time—10 or 11 days)

m217c-positive cells in neural crest (NC) cultured for 6 or 7 days and then in presence or absence of E16 retina for 4 days (total NC culture time—10 or 11 days)
m217c-positive cells in neural crest (NC) cultured for 6 or 7 days and then in presence or absence of E16 retina for 4 days (total NC culture time—10 or 11 days)
Fig. 2.

(A) Graph of bipolar cells vs distance from point A in neural crest cultured for 6 or 7 days and then in presence or absence of E16 retina for 4 days (total NC culture time=10 days). A=a central point on the edge of the retina explant, or in controls, a point in the NC outgrowth similar to where the retina would be put in co-cultures, identified on day 6 to 7 and referred back to on day 10 or 11 after fixation and staining. The vertical bar represents 2 standard errors of measurement derived from the interaction term of the analysis of variance. (B) Graph of m217c stained cells vs distance from point A in neural crest cultured for 6 or 7 days and then in presence or absence of E16 retina for 4 days. (C) Graph of bipolar cells vs distance from point A in NC cultured for 5 to 7 days and then cultured in the presence or absence of E16 retina or in astrocyte-conditioned medium for 6 or 7 days (total NC culture time=12 or 13 days). (D) S-100-containing cells in neural crest cultured for 5 to 7 days and then cultured in the presence or absence of E16 retina or in astrocyte conditioned medium for 6 or 7 days (total NC culture time=12 or 13 days).

Fig. 2.

(A) Graph of bipolar cells vs distance from point A in neural crest cultured for 6 or 7 days and then in presence or absence of E16 retina for 4 days (total NC culture time=10 days). A=a central point on the edge of the retina explant, or in controls, a point in the NC outgrowth similar to where the retina would be put in co-cultures, identified on day 6 to 7 and referred back to on day 10 or 11 after fixation and staining. The vertical bar represents 2 standard errors of measurement derived from the interaction term of the analysis of variance. (B) Graph of m217c stained cells vs distance from point A in neural crest cultured for 6 or 7 days and then in presence or absence of E16 retina for 4 days. (C) Graph of bipolar cells vs distance from point A in NC cultured for 5 to 7 days and then cultured in the presence or absence of E16 retina or in astrocyte-conditioned medium for 6 or 7 days (total NC culture time=12 or 13 days). (D) S-100-containing cells in neural crest cultured for 5 to 7 days and then cultured in the presence or absence of E16 retina or in astrocyte conditioned medium for 6 or 7 days (total NC culture time=12 or 13 days).

6 days after addition of retina to NC cultures, they were stained for S-100 and RT97 anti-neurofilament. RT97 labelled the profuse outgrowth of retinal neurites, which extended over the neural crest cells for considerable distances up to 3500 pm from the retinal explant. Large numbers of S-100-positive bipolar cells were observed in the vicinity of the retina in the area where there were neurites (Fig. 3A,B), and many of these aligned and co-localised with the fascicles (Fig. 3C,D). However, not all the bipolar cells contained S-100. S-100-stained cells were generally near but not always in contact with retinal neurons; many S-100-positive cells were observed up to 200 μm away from retinal neurons. Not all regions of the culture in contact with axons turned into Schwann cells; in some areas near the retina, where there was an extensive network of axons, no S-100-containing cells were present (see Fig. 3C,D). Cell counts and t-tests revealed that there were significantly more S-100-stained cells and bipolar cells at a distance of 0 to 1000 μm from A, a point on the edge of the retina than in a similar position in control cultures (see Tables 3 and 4 and Fig. 2C,D). When a two-factor analysis of variance was conducted, the effect of the retina on bipolar morphology was highly significant compared to either neural crest cultured alone or in astrocyte-conditioned medium (main effect due to culture type, F=6.11 with 2,10 df, P=<0.018; main effect due to distance from A F=9.20 with 4,40 df, P= <0.001; culture type x distance interaction, F=3.05 with 8,40df P=<0.009). The effect of retina on S-100 protein accumulation was highly significant compared to neural crest cultured alone or in astrocyte-conditioned medium (main effect due to culture type, F=7.73 with 2,14df, P<0.005; main effect due to distance from A F=8.89 with 4,49 df, P<0.001; culture typexdistance interaction, F=4.93 with 8,49df, P<0.001). At a distance from the retinal explant and retinal neurite outgrowth, isolated or small clusters of neurons were present which had presumably differentiated from neural crest cells, and these were also observed in control cultures. Varying numbers of scattered S-100-containing cells were associated with these patches, and these were near but not always in contact with either neurites or neuronal cell bodies (Fig. 4A,B). When cultures were double stained for m217c and S-100 all S-100-stained cells were also positive for m217c.

Table 3.

Bipolar cells in neural crest cultured for 5 to 7 days and then cultured in the presence or absence of E16 retina or in astrocyte-conditioned medium (CM) for 6 or 7 days (total NC culture time=12 or 13 days)

Bipolar cells in neural crest cultured for 5 to 7 days and then cultured in the presence or absence of E16 retina or in astrocyte-conditioned medium (CM) for 6 or 7 days (total NC culture time=12 or 13 days)
Bipolar cells in neural crest cultured for 5 to 7 days and then cultured in the presence or absence of E16 retina or in astrocyte-conditioned medium (CM) for 6 or 7 days (total NC culture time=12 or 13 days)
Table 4.

S-100-positive cells in neural crest cultured for 5 to 7 days and then cultured in the presence or absence of E16 retina or in astrocyte-conditioned medium (CM) for 6 or 7 days (total NC culture time=12 or 13 days)

S-100-positive cells in neural crest cultured for 5 to 7 days and then cultured in the presence or absence of E16 retina or in astrocyte-conditioned medium (CM) for 6 or 7 days (total NC culture time=12 or 13 days)
S-100-positive cells in neural crest cultured for 5 to 7 days and then cultured in the presence or absence of E16 retina or in astrocyte-conditioned medium (CM) for 6 or 7 days (total NC culture time=12 or 13 days)
Fig. 3.

(A) Neural crest cultured for 6 days and then co-cultured with E16 retina (r) for 7 days. Culture fixed and double stained with antibody to S-100 protein and mAb RT97 to neurofilament protein. Large numbers of S-100 containing bipolar cells (arrows) are in the vicinity of the axons (see Fig. 3B). Bar=100μm. (B) Same field as (A). Retinal axons are stained with antiserum RT97 to neurofilament protein. r=retinal expiant. Bar=100μm. (C) Neural crest cultured for 5 days and then co-cultured with E16 retina for 7 days. Culture fixed and stained with antibody to S-100 protein. Large numbers of S-100-stained bipolar cells (arrows) are localised with a fascicle of retinal neurites (see D). Bar=100μm. (D) Same field as C. Retinal axons are stained with mAb RT97 to neurofilament protein. Rt97 also stained the neural crest cell nuclei. Bar=100μm.

Fig. 3.

(A) Neural crest cultured for 6 days and then co-cultured with E16 retina (r) for 7 days. Culture fixed and double stained with antibody to S-100 protein and mAb RT97 to neurofilament protein. Large numbers of S-100 containing bipolar cells (arrows) are in the vicinity of the axons (see Fig. 3B). Bar=100μm. (B) Same field as (A). Retinal axons are stained with antiserum RT97 to neurofilament protein. r=retinal expiant. Bar=100μm. (C) Neural crest cultured for 5 days and then co-cultured with E16 retina for 7 days. Culture fixed and stained with antibody to S-100 protein. Large numbers of S-100-stained bipolar cells (arrows) are localised with a fascicle of retinal neurites (see D). Bar=100μm. (D) Same field as C. Retinal axons are stained with mAb RT97 to neurofilament protein. Rt97 also stained the neural crest cell nuclei. Bar=100μm.

Fig. 4.

(A) Neural crest cultured for 5 days and then cocultured with E16 retina for 7 days. Culture fixed and double stained with antibody to S-100 protein and mAb RT97 to neurofilament protein. S-100-stained bipolar cells are near but not in contact with a neurite (see B). (This neuron was at a distance from the retina and had probably differentiated from a neural crest cell). Bar=50μm. (B) Same field as A. Neurite stained with mAb RT97 to neurofilament protein. Bar=50μm.

Fig. 4.

(A) Neural crest cultured for 5 days and then cocultured with E16 retina for 7 days. Culture fixed and double stained with antibody to S-100 protein and mAb RT97 to neurofilament protein. S-100-stained bipolar cells are near but not in contact with a neurite (see B). (This neuron was at a distance from the retina and had probably differentiated from a neural crest cell). Bar=50μm. (B) Same field as A. Neurite stained with mAb RT97 to neurofilament protein. Bar=50μm.

In order to be sure that the S-100 stained bipolar cells in our cultures were derived from neural crest cells and had not migrated from the explant, we labelled 3 retinal expiants with the dye CFSE (carboxyfluorescein succi-dimidyl ester), which fluorescently labelled the cells of the explant, and co-cultured these expiants on top of neural crest cells. When observed 2 and 7 days later, there were only a few small round cells that had migrated from the explant, and there were many bipolar cells that were not fluorescently labelled (Fig. 5A,B).

Fig. 5.

(A) Neural crest cultured for 6 days and then with E16 retina labelled with CFSE for 2 days. Phase contrast. Bar=200μm. (B) Neural crest cultured for 6 days and then with E16 retina labelled with CFSE for 2 days. Fluorescence. Bar=200μm.

Fig. 5.

(A) Neural crest cultured for 6 days and then with E16 retina labelled with CFSE for 2 days. Phase contrast. Bar=200μm. (B) Neural crest cultured for 6 days and then with E16 retina labelled with CFSE for 2 days. Fluorescence. Bar=200μm.

Retinal expiants contain neurons, and also a few glial cells. In order to see whether the retinal glial cells might have been secreting factors that induced Schwann cell differentiation, we added astrocyte-conditioned medium to 5 day neural crest cultures, and stained them for S-100 7 days later. The number of bipolar cells and S-100-stained cells was no different from controls in these cultures (see Tables 3 and 4 and Fig. 2C,D). We conclude that the neuronal cells of the expiants are responsible for inducing Schwann cell differentiation.

Staining controls

No immunoreactivity was observed when the primary antibody was omitted from the staining procedure or when it was replaced with rabbit serum. Other monoclonal antibodies from the same immunoglobulin class as m217c give different staining patterns. These control antibodies are 151-IgG, a mAb that recognises the rat EGF receptor (Parsons Chandler et al. 1985) and anti GFAP (Boehringer). The third control class matched mAb was F16.4.4, an antibody to Class 1 antigen of the rat major histocompatibility complex (Hart and Fabre, 1981; Jeff Butcher, personal communication). This antibody recognised many cells in sections of rat liver but failed to recognise any cells in rat neural crest cultured for 3 days. The binding pattern of antibodies to S-100 protein and laminin antisera were also unique to each of these antisera.

Our results demonstrate that the presence of retinal neurons causes NC cells to differentiate into Schwann cells. The first stage of differentiation occurred within 4 days of addition of the retina to the neural crest; retinal axons had by this time extended over the neural crest cells, and we observed that large numbers of neural crest cells adjacent to and in the vicinity of the axons had acquired a bipolar morphology typical of Schwann cells. Next, and within 6 days of co-culture, there was a significant increase in the number of cells expressing the relatively mature Schwann cell marker, S-100; as with bipolar morphology this increase was only significant within 1000nm of the explant. In addition, localized high density patches of S-100-containing cells were also sometimes observed next to fascicles of retinal neurites that extended for distances up to 3500μm from the retinal explant. Many of the bipolar and S-100-stained cells were aligned along retinal axons, but they were not always in contact with neuronal processes, we saw many cells that were up to 200 μm from the nearest axon. The effect of neurons on NC cell differentiation may thus be mediated by a diffusible compound, but one secreted in relatively small amounts, so as to affect only cells fairly close to axons: alternatively it is possible that some of the differentiated Schwann cells had contacted axons and subsequently migrated away. Despite increasing the numbers of S-100-containing and bipolar cells, the addition of retinal expiants to neural crest cultures had no effect on the overall number of cells that stained with m217c. This antibody stains a proportion of neural crest cells from the earliest stages of culture, and in the premigratory population in vivo, and therefore presumably marks cells that have achieved only the very first stages of the differentiative pathway that leads to Schwann cells. Our results are consistent with the notion that the diffusible factor secreted by neurons is only active on cells that have already started to express the antigen for m217c, and have therefore made the first steps towards becoming Schwann cells. In theory some of our results could be explained by an effect on Schwann cell survival. We think this is unlikely, since we saw little cell death in our cultures, and since the size of the 217c-stained cell population, which includes the Schwann cells, did not change between experimentáis and controls.

In this experiment, as in many others, neurons of central nervous system origin have a similar effect on Schwann cells to those of peripheral nervous system origin. For instance, CNS axons will regenerate into Schwann cell containing peripheral nerve grafts, and will be myelinated by Schwann cells there (David and Aguayo, 1981), and both CNS and PNS neuronal membranes are mitogens for Schwann cells (Mason et al. 1989; Salzer et al. 1980). On the other hand, enteric neurons appear to have a negative mitogenic effect (Eccleston et al. 1989). In the present experiment, both the axons growing from the retinal explant and those that had differentiated from neural crest cells in the culture were surrounded by Schwann cells. The factor that induces Schwann cell differentiation must, therefore, be common to both CNS and PNS.

Retinal neurites grew rapidly and for considerable distances on neural crest cells. It is likely that this outgrowth is at least in part attributable to the presence of laminin, which is present on the neural crest cells. Laminin has been shown to strongly promote neurite extension from both CNS and PNS neurons (Baron von Evercooren et al. 1982; Manthorpe et al. 1983) and it has previously been observed that rat E16 retina (Cohen and Johnson, 1990) and early embryonic chick retina (Cohen et al. 1986) are dependent on LN for neurite extension.

Our results demonstrate that neural crest cells require a close association with neurons in order to differentiate into Schwann cells. Since cultured crest cells do not encounter the tissues found on their normal migratory pathway, it is likely that neural crest cells do not require a prior interaction with these other tissues (e.g. notochord and somites) in order to respond to neurons, although it is possible that factors in the culture medium could have provided some such clues. Previous observations have suggested that Schwann cells probably differentiate from a subpopulation of neural crest cells that differentiate into Schwann cells or PNS neurons prior to leaving the neural tube or shortly after migration commences (Smith-Thomas and Fawcett, 1989), and the present experiment shows that the presence or absence of neurons does not affect the rate at which this first differentiative step occurs. The observation that many Schwann cells that differentiate from neural crest cells are not directly in contact with neurons or their processes suggests that, unlike the later stages of Schwann cell differentiation (Bunge, 1987; Jessen et al. 1987), continuous contact between neural crest cell and neuronal membranes is not essential for these earlier stages of Schwann cell differentiation; instead, a short-range diffusible compound is probably involved.

This work was supported by the Medical Research Council (UK). We thank Peter Starling for help with the photography and Dr Steve Dunnett for help with the statistics.

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