Embryonic (9-day) chick neuroretinal cells transdifferentiate extensively into lens and pigment cells during prolonged culture (4–5 weeks) in media containing foetal calf serum. Medium conditions which promote the attachment and differentiation of neural cells in other culture systems (e.g. horse serum, high glucose levels) both delay the onset and greatly reduce the extent of transdifferentiation in retinal cultures. In the presence of high glucose, horse serum (but not foetal calf serum) also favours cholinergic neuronal differentiation during the early phases of culture, as shown by the levels of choline acetyltransferase activity and accumulation of labelled choline. Substrate conditions have some effect on cholinergic differentiation (promoted by polylysine-coated dishes) but do not affect later transdifferentiation. These effects may be due in part to selective survival or growth of particular retinal cell types under the various medium conditions tested. Cultures stripped of neuronal cells contain negligible choline acetyltransferase activity, but still transdifferentiate into both lens and pigment cells, although more slowly than control cultures. Cell size distributions reveal a significant depletion of the larger cells in high glucose media with foetal calf serum, but not in those with horse serum.

The term metaplasia is used to describe the conversion of a cell from one fully-differentiated phenotype to another, a process involving dedifferentiation followed by redifferentiation (as exemplified by Wolffian lens regeneration from the dorsal iris epithelium in newts; reviewed by Yamada, 1977). Transdifferentiation, by contrast, is a term of wider application, used to describe the conversion of partially-differentiated cells from one tissue into fully-differentiated cells of a foreign tissue type (see Okada, 1976). The appearance of lens and pigment cells during long-term culture of embryonic chick neural retina (Okada, Itoh, Watanabe & Eguchi, 1975; Itoh, Okada, Ide & Eguchi, 1975) is of this type, since the initial tissue is not yet fully differentiated, although clearly neither lens nor pigment cells would ever appear among normal neuroretinal cells in vivo. This capacity for transdifferentiation during long-term culture in vitro may reflect some relict potential of the optic cup tissues to regenerate both lens (as in Wolffian lens regeneration) and each other (Lopashov, 1963; Coulombre & Coulombre, 1965). In vivo however the neural retina develops into a multilayered neural tissue comprising supportive Müller cells (glia) and several morphologically distinctive neuronal cell types (photoreceptor cells, bipolar and horizontal cells, amacrine cells and ganglion cells; reviewed by Cajal, 1893). These layers become established by about the 15th day of embryonic development in chick, at which stage transdifferentiation into lens in culture is still possible, though much less pronounced than at earlier stages (de Pomerai & Clayton, 1978; Nomura & Okada, 1979). The various cell layers of the avian retina utilize a number of different neurotransmitter systems, including acetylcholine (Vogel & Nirenberg, 1976; Cristani-Combes, Pessac & Calothy, 1978), catecholamines (Ehinger, 1967), GABA (Marshall & Voaden, 1974) and possibly also substance P (Karten & Brecha, 1979) and serotonin (Suzuki, Noguchi & Yagi, 1978).

In cultures of -day embryonic chick neuroretinal cells, there is an initial increase in choline acetyltransferase (CAT; E.C.2.3.1.6) activity, followed by a sharp decline when 8 crystallin begins to appear (Okada, Yasuda & Nomura, 1979). CAT is a marker of cholinergic neuronal differentiation in chick retina (Crisanti-Combes et al. 1978), while δ crystallin is confined to chick lens fibres laid down during embryonic and early post hatching development (Clayton, 1974, 1979). The present paper extends this approach, using cultures of neuroretinal cells from later 9-day chick embryos, and examining two parameters of cholinergic neuronal differentiation (CAT activity and choline accumulation) under culture conditions used widely in other neural systems to encourage neuronal differentiation. These include the use of horse serum (Fedoroff & Hall, 1979) in combination with high glucose levels (Adler, Manthorpe & Varón, 1979), and highly adhesive substrates such as polylysine-coated dishes (Pettmann, Louis & Sensenbrenner, 1979). No combination of these conditions yet tested can block transdifferentiation completely, though media based on horse serum both delay its onset and greatly reduce its extent, while favouring various aspects of cholinergic neuronal differentiation during the earlier phases of culture. The effects of these conditions on other aspects of retinal differentiation (including markers for other neurotransmitter systems and for Müller cells) in cultures of 9-day and later embryonic neuroretinal cells, are under current investigation.

Fertile chick eggs were obtained from J. B. Eastwood Ltd (Nottingham). Sources of chemicals and apparatus are given below where appropriate.

Cell culture

Neuroretinal cells from 9-day chick embryos were cultured as described previously (Okada et al. 1975; de Pomerai, et al. 1977; de Pomerai & Clayton, 1978), except that retinas were dissociated for 60 min. in 0-002% Trypsin (Sigma Type 1) in calciumand magnesium-free saline (CMF) to minimize damage to neuronal cells (Varón & Raiborn, 1969). Standard culture medium F was Eagle’s MEM with Earle’s salts and 2 mM L-glutamine, supplemented with 10% foetal calf serum (FCS), 100 units/ml Penicillin,, 100/ig/ml Streptomycin (all from GIBCO - Europe, Paisley, Scotland) and 26 mM NaHCO3. H medium contained 10% horse serum (GIBCO) in place of FCS. Hg and Fg media were H and F media containing extra glucose to 28 mM (insteaed of 6 mM) final. Some dishes were coated with polylysine (5 μg/ml; M.W. >70000; Sigma Type 1B) according to Pettmann et al. 1979. Certain cultures were largely (> 90%) stripped of neuronal cells after 4 days in vitro by gentle swirling following a 15-min incubation in 0·001% Trypsin/O-Cl% collagenase (type 1, Sigma) in CMF (K. Yasuda, personal communication). All cultures were seeded with 3-7 x 10® neuroretinal cells per ml of culture medium, and were maintained in vitro for up to 8 weeks as described previously (de Pomerai et al. 1977), the medium being changed every 2·3 days. Cell counting after trypsinisation (0-2% Sigma Type 1 in CMF for 2-3 h) and photography of cultures were also as described previously (de Pomerai et al. 1977). A model ZB1 Coulter counter was used to obtain cell size distributions (cell suspension diluted 1·5 ml to 30 ml) at 1 μm diameter intervals across the size range 1·25 μm. The counter was calibrated using 18·5 μm diameter Latex beads (Coulter Ltd).

Identification of crystallins

Analysis of the soluble proteins extracted from cultures was performed by SDS-polyacrylamide gradient slab gel electrophoresis, as detailed previously (de Pomerai & Clayton, 1978), and protein contents were determined by the Lowry method (Lowry, Rosebrough, Farr & Randall, 1951). Crystallins were identified by comparison with chick lens protein standards (Thomson et al. 1978).

Haemagglutination inhibition assays were performed as described previously (de Pomerai & Clayton, 1978,1980), except that the marker sheep red blood cells were coated with a mixture of equal parts of soluble proteins from newly-hatched chick lens (rich in 8 crystallin) and from adult chick lens cortex (rich in a and ft crystallins) by the method of Evans, Steel and Arthur (1974). This gave good agglutination with the anti-α, anti-total-β and anti-δ crystalline-specific antisera, which were the same as described previously (de Pomerai & Clayton, 1978, 1980). Assays were always performed in duplicate, and several sets of such assay results have been averaged to give a final mean.

Assessment of neuronal differentiation

(a) Choline acetyltransferase (CAT) assays,

These assays were performed on culture extracts as detailed by Crisanti-Combes et al. (1978), except that all assay components except [14C] Acetyl-Co A (59μCiμmole; Radiochemical Centre, Amersham) were present at one fifth of the specified final concentrations. Blank assay values (using extraction buffer in place of culture extract) have been subtracted from all experimental results. All assays were performed in duplicate, and several sets of data from a number of independent culture batches have been averaged to give the means and standard errors plotted in Fig. 7.

(b) Choline accumulation

Choline incorporation into membrane components was assessed by the method of Baraid & Berg (1979). Cultures were washed and incubated in 1 μM-[3H]choline (55 Ci/mmole; Radiochemical Centre, Amersham) in phosphate-buffered saline (PBS) for one hour, followed by extensive washing for a further hour in PBS containing 10 μM unlabelled choline. Hemicholinium III (10 μM) was present throughout the procedure in some cases, so as to inhibit specific choline uptake (Baraid & Berg, 1979).

A Growth and morphology

Of the four culture media originally tested (see Fig. 1), that with horse serum but no added glucose (H) failed to support cell growth and was not further studied. During the first two weeks of culture, cell numbers fall progressively under all conditions, but more markedly in media containing foetal calf serum (F, Fg) as compared to horse serum (H, Hg) and independent of added glucose (Fig. 1). When cell numbers start to recover, however, unsupplemented F medium supports significantly faster culture growth, whereas cell numbers increase more slowly in the glucose-supplemented media, independent of the serum type (Hg and Fg; Fig. 1). This suggests that the neuronal and glial cell populations present in retinal cultures (Okada et al. 1979) may respond differently to these various medium conditions in terms of survival and/or growth. Further indications of this are given in Fig. 2, which shows a series of cell size distributions obtained from freshly-dissociated 9-day embryo retinas and from cultures of the same cell batch maintained for 4, 10 and 20 days in F, Fg and Hg media. Of particular interest at 10 and 20 days is the tail-out towards larger cell sizes (10–16μm diameter) prominent in F and especially Hg cultures (relative to the numbers of smaller cells) but severely depleted in Fg medium. It should be noted that the largest ( > 12 μm) class of cells initially present in fresh retina almost disappears by 4 days in culture.

Fig. 1

Growth of 9-day embryonic neuroretinal cultures in F, Fg, H and Hg media. Cultures were set up and maintained in these four media as described in Methods. At the indicated time points duplicate dishes were trypsinized and the cells counted by haemocytometer. At least 2000 cells were counted per sample, and duplicate dishes varied by less than 10% from each other. Mean cell counts from both dishes have been plotted without ranges for the sake of clarity. ▪ —▪, F medium; ▫ — — — ▫, Fg medium; ○ – – – –○, H medium; ● …….●, Hg medium.

Fig. 1

Growth of 9-day embryonic neuroretinal cultures in F, Fg, H and Hg media. Cultures were set up and maintained in these four media as described in Methods. At the indicated time points duplicate dishes were trypsinized and the cells counted by haemocytometer. At least 2000 cells were counted per sample, and duplicate dishes varied by less than 10% from each other. Mean cell counts from both dishes have been plotted without ranges for the sake of clarity. ▪ —▪, F medium; ▫ — — — ▫, Fg medium; ○ – – – –○, H medium; ● …….●, Hg medium.

Fig. 2

Cell size distributions for retinal cultures in F, Fg and Hg media. These cell size distributions were obtained using a Model ZB1 Coulter counter with appropriate threshold settings for 1 μm diameter intervals in the range 1-25 μm. The four parts of Fig. 2 were derived from a single experiment, the cells (T5 ml suspension diluted to 30 ml) from one dish of each type being counted (0·1 ml per count) at each stage. Part A, freshly dissociated retina. Part B, retinal cultures at 4 days. Part C, retinal cultures at 10 days. Part D, retinal cultures at 20 days. For parts B, C, and D, histogram bars are identified as follows: —F medium;, Fg medium – – –Hg medium. Similar assessments have been performed with three independent sets of cultures, the overall size distributions in the three media being virtually indistinguishable from that shown. N.B. The histograms plotted in this figure give actual cell numbers within the indicated size ranges from 01 ml samples of the diluted suspension; no attempt has been made to correct for the different numbers of cells present under .the three medium conditions (see Fig. 1).

Fig. 2

Cell size distributions for retinal cultures in F, Fg and Hg media. These cell size distributions were obtained using a Model ZB1 Coulter counter with appropriate threshold settings for 1 μm diameter intervals in the range 1-25 μm. The four parts of Fig. 2 were derived from a single experiment, the cells (T5 ml suspension diluted to 30 ml) from one dish of each type being counted (0·1 ml per count) at each stage. Part A, freshly dissociated retina. Part B, retinal cultures at 4 days. Part C, retinal cultures at 10 days. Part D, retinal cultures at 20 days. For parts B, C, and D, histogram bars are identified as follows: —F medium;, Fg medium – – –Hg medium. Similar assessments have been performed with three independent sets of cultures, the overall size distributions in the three media being virtually indistinguishable from that shown. N.B. The histograms plotted in this figure give actual cell numbers within the indicated size ranges from 01 ml samples of the diluted suspension; no attempt has been made to correct for the different numbers of cells present under .the three medium conditions (see Fig. 1).

Figure 3 compares the development of retina cultures in F, Fg and Hg media on plastic dishes, while Fig. 4 compares development in F and Hg media on polylysine-coated dishes. In morphological terms, neuronal differentiation is more apparent in Hg and Fg media as compared to F medium at 15 days (Fig. 3, i-iii); cell processes are also better developed on a polylysine substrate in Hg as compared to F medium (Fig. 4, i-iv; at 6 and 15 days). By 40 days, lentoids (aggregates of lens fibre cells wrapped around each other; Okada et al. 1975) and pigment cells are prominent in all cultures maintained in F medium, (Fig. 3, iv), but few if any could be found in those kept in Hg medium (Fig. 3, vi). This remains true even after 60 days of culture in Hg medium (results not shown). Some lentoids and pigment cells also develop in Fg cultures after about 40 days in vitro (Fig. 3, v), though less extensively than in F medium (Fig. 3, iv). Cells of neuronal morphology, however, can survive in Hg and Fg cultures for periods of 40 to 50 days (Fig. 3, v, and vi), whereas most such cells disappear from F cultures after about 30 days (Fig. 3, iv; c.f. Okada et al. 1975; de Pomerai et al. 1977). The contrast between F and Hg media in terms of lentoid formation does not seem to be affected by the culture substrate, as shown by parallel cultures on polylysine-coated dishes (Fig. 4, v and vi; at 40 days).

Fig. 3

Development of retinal cultures in F, Fg and Hg media. This figure shows typical fields from living cultures on plastic dishes photographed under phase contrast. Top row, cultures at 15 days. Bottom row, cultures at 40 days. Superscripts F, Fg and Hg denote the media used. Bar represents 100 μm.

Fig. 3

Development of retinal cultures in F, Fg and Hg media. This figure shows typical fields from living cultures on plastic dishes photographed under phase contrast. Top row, cultures at 15 days. Bottom row, cultures at 40 days. Superscripts F, Fg and Hg denote the media used. Bar represents 100 μm.

Fig. 4

Effect of substrate on retinal culture differentiation in F and Hg media. These fields were photographed from living cultures maintained in F (first column) and Hg (second column) media on polylysine-coated plastic dishes. Top row, cultures at 6 days; middle row, cultures at 15 days; bottom row, cultures at 40 days. Bar represents 100μm.

Fig. 4

Effect of substrate on retinal culture differentiation in F and Hg media. These fields were photographed from living cultures maintained in F (first column) and Hg (second column) media on polylysine-coated plastic dishes. Top row, cultures at 6 days; middle row, cultures at 15 days; bottom row, cultures at 40 days. Bar represents 100μm.

B Crystallin appearance

Figures 5 and 6 demonstrate the appearance of chick lens crystallins in these cultures, using haemagglutination inhibition assays with specific anticrystallin antisera (Fig. 5) and SDS polyacrylamide gradient slab gel electrophoresis (Fig. 6). The crystallins (especially δ) appear in much greater amounts in cultures maintained in F medium as compared to Hg medium (α+ β + δ crystallin represents some 40% of the total soluble protein extracted from 52-day-old cultures in F medium, as compared to 3% in Hg medium at the same stage; Fig. 5, A and C). Cultures in Fg medium tend to degenerate after about 50 days in vitro, and reliable data were not obtained beyond 46 days. Nevertheless, it is clear that this medium gives intermediate results throughout (Fig. 5B). /? crystallin is more prominent than δ for cultures in Hg medium, whereas the converse is true for Fg and particularly F media. This is in accord with previous results showing that α + β: δcrystallin ratio increases with declining extent of transdifferentia-tion, whether due to advancing embryonic age (de Pomerai & Clayton, 1978) or reduced culture growth rate (de Pomerai & Clayton, 1980). Table IB compares the amounts of crystallins after 34 and 50 days in vitro in controls and in retinal cultures which had been stripped of at least 90% of their neuronal cells (de Pomerai and Gali, unpublished data). Both lentoids and pigment cells appear in these ‘epithelial ‘cultures, although more slowly than in controls. This is true for all three classes of crystallin, the levels being four-to eightfold lower than controls at 34 days, but at most twofold lower by 50 days. Again crystallin is more prominent in epithelial cultures than in controls ( levels at 50 days are almost identical). This is compatible with the suggestion (de Pomerai & Clayton, 1980) that neuronal cells may contribute (directly or indirectly) a population of l-rich lentoids during transdifferentiation, although other explanations (e.g. in terms of cell density) have not been tested yet.

Fig. 5

Appearance of crystallins in retinal cultures under various conditions. This figure shows the results of haemagglutination inhibition assays performed on the soluble proteins extracted from 20- to 52-day-old cultures of 9-day embryonic chick neuroretinal cells (see Methods). Parts A, B and C compare the appearance of crystallins in these cultures under three medium conditions, namely Hg (part A), Fg (part B) and F (part C). At least four sets of duplicate assay results were used in compiling these figures, the mean and standard error (vertical bar) being plotted for all points except that at 20 days (when standard error bars lie within the symbol area). ▴ —▴, δcrystallin; ● ……●, β crystallins; ▪ – – – – – –▪ α crystallin.

Fig. 5

Appearance of crystallins in retinal cultures under various conditions. This figure shows the results of haemagglutination inhibition assays performed on the soluble proteins extracted from 20- to 52-day-old cultures of 9-day embryonic chick neuroretinal cells (see Methods). Parts A, B and C compare the appearance of crystallins in these cultures under three medium conditions, namely Hg (part A), Fg (part B) and F (part C). At least four sets of duplicate assay results were used in compiling these figures, the mean and standard error (vertical bar) being plotted for all points except that at 20 days (when standard error bars lie within the symbol area). ▴ —▴, δcrystallin; ● ……●, β crystallins; ▪ – – – – – –▪ α crystallin.

Fig. 6

Appearance of crystallins in retinal cultures maintained in F, Fg and Hg media. Soluble proteins were extracted from F and Fg cultures at 25 and 35 days, and 100 μg samples run on an SDS-polyacrylamide gradient slab gel as described in Methods. Slot 1: F cultures after 25 days. Slot 2: Fg cultures after 25 days. Slot 3: F cultuies after 35 days. Slot 4: Fg cultures after 35 days. Slot 5: 50μg of newly-hatched chick lens soluble protein. 80 μg samples of soluble protein from 36 day cultures maintained in F and Hg media were run as above on a separate gel. Slot 6: 75 μg of newly-hatched chick lens soluble protein. Slot 7: Hg cultures after 36 days. Slot 8: F cultures after 36 days. Crystallin bands identified according to Thomson et al. (1978).

Fig. 6

Appearance of crystallins in retinal cultures maintained in F, Fg and Hg media. Soluble proteins were extracted from F and Fg cultures at 25 and 35 days, and 100 μg samples run on an SDS-polyacrylamide gradient slab gel as described in Methods. Slot 1: F cultures after 25 days. Slot 2: Fg cultures after 25 days. Slot 3: F cultuies after 35 days. Slot 4: Fg cultures after 35 days. Slot 5: 50μg of newly-hatched chick lens soluble protein. 80 μg samples of soluble protein from 36 day cultures maintained in F and Hg media were run as above on a separate gel. Slot 6: 75 μg of newly-hatched chick lens soluble protein. Slot 7: Hg cultures after 36 days. Slot 8: F cultures after 36 days. Crystallin bands identified according to Thomson et al. (1978).

Fig. 7

Choline acetyltransferase activity in F, Fg and Hg cultures. CAT assays were performed (as described in Methods) at various stages during cell culture in the three media, and results are given as net dpm (above blank values) per μ g soluble protein. Mean and standard error (vertical bar) are plotted for all points where three or more independent cultures (each sample in duplicate) have been assayed. Some additional points are included where only one or two cultures have been assayed (in duplicate); these are plotted as single mean points and should be regarded as merely indicative. The data plotted in this figure were obtained from several different sets of cultures set up at initial cell densities ranging from 3 to 7 × 106 cell per ml. Part A, F medium; part B, Fg medium; part C, Hg medium.

Fig. 7

Choline acetyltransferase activity in F, Fg and Hg cultures. CAT assays were performed (as described in Methods) at various stages during cell culture in the three media, and results are given as net dpm (above blank values) per μ g soluble protein. Mean and standard error (vertical bar) are plotted for all points where three or more independent cultures (each sample in duplicate) have been assayed. Some additional points are included where only one or two cultures have been assayed (in duplicate); these are plotted as single mean points and should be regarded as merely indicative. The data plotted in this figure were obtained from several different sets of cultures set up at initial cell densities ranging from 3 to 7 × 106 cell per ml. Part A, F medium; part B, Fg medium; part C, Hg medium.

In Fig. 6, the prominence of protein bands migrating to the same positions as the a and 8 crystallins correlates well with the results shown for F, Fg and Hg cultures in Fig. 5 (A, B, and C). After 36 days in vitro, these proteins are more prominent in F than in Fg cultures and are virtually absent in Hg cultures. However, a minor retinal protein of the same apparent subunit molecular weight as δ crystallin is present in Hg cultures and also at 25 days in F and Fg cultures; in all these cases, δ crystallin is barely detectable immunologically (c.f. de Pomerai et al. 1977; de Pomerai & Clayton, 1978). Soluble proteins were also compared between F and Hg media for 36 day cultures on plastic, collagen or polylysine substrates (results not shown); few if any differences according to substrate were found, although the pattern of protein bands was consistently different in Hg as compared to F cultures. In particular, the prominent bands found at the a and ô crystallin positions in all F culture samples were much reduced or absent in all Hg culture samples (c.f. Fig. 6, Hg and F at 36 days). Overall, Fg medium reduces lentoid and crystallin production relative to F medium, while Hg medium largely eliminates both. Substrate conditions have little effect on this contrast.

C Choline metabolism

Figure 7 shows the results of choline acetyltransferase (CAT) assays using cultures grown under all three medium conditions. After an initial two-to threefold increase above the levels found in fresh 9-day embryo retinal cells, there is a decline in CAT activity in F and most markedly in Fg cultures, whereas in Hg cultures the CAT activity reaches higher levels and is maintained for considerably longer. After about 20 days, CAT activity is negligible in all types of retinal culture studied to date (de Pomerai, unpublished data). It should be noted that the data included in Fig. 7 are derived from several different sets of cultures (not merely that used for Fig. 1) and that the differences in CAT activity (per μg protein) were maintained even between low-density Hg cultures and high-density F cultures. A non-specific cell-density effect on CAT activity is thus unlikely to explain these results (see also Discussion). Substrate conditions have little effect during the first 8 days of culture, but polylysine-coated dishes promote CAT activity markedly between 8 and 16 days in vitro, as shown in Table 1A (at 12 days). Conversely, cultures which have been largely ( > 90%) stripped of neuronal cells after 4 days in vitro, contain negligible CAT activity at 12 days (Table 1 A). Overall, CAT activity correlates well with the morphological development of neuronal cells under F and Hg (but not Fg) conditions during the first 2 weeks of culture (c.f. Figs. 3 and 4). Thereafter, the surviving cells of neuronal morphology lack significant CAT activity, even though they can apparently still bind the neurone-specific marker tetanus toxin (Mirsky et al. 1979), as revealed by indirect immunofluorescence (de Pomerai, unpublished results).

Table 1a

Choline acetyltransferase activity in 12-day cultures of retinal cells

Choline acetyltransferase activity in 12-day cultures of retinal cells
Choline acetyltransferase activity in 12-day cultures of retinal cells
Table 1 b

Crystallin contents of control and epithelial cultures

Crystallin contents of control and epithelial cultures
Crystallin contents of control and epithelial cultures

The trend shown by Fig. 7 is supported by preliminary studies of [3H]choline accumulation. For cultures at 8 days, the rate of choline uptake (over a 5 to 60 minute assay period) is consistently higher for Hg than for F medium across the concentration range 10−8 to 10−6 M choline, and at these low concentrations, choline is apparently taken up against a concentration gradient (de Pomerai, unpublished data). Further studies of the kinetics and sodium and temperature dependence of this process are now under way.

For Table 2, retinal cultures were labelled for 1 h in 1 μM [3H]choline and then washed for a further hour in 10 μM unlabelled choline. According to Barald and Berg (1979) this technique detects mainly the choline incorporated into membrane components. For Hg cultures at 8 days, the rate of choline uptake (per unit protein) immediately after labelling is higher than for F cultures (see above), yet choline conversion into membrane material is markedly lower at 3 and 10 days for Hg as compared to F or Fg cultures (Table 2). This might suggest that a larger fraction of the choline taken up by Hg cultures may be metabolised e.g. as acetylcholine, consistent with the higher CAT activity in these cultures (Fig. 7). Hemicholinium III inhibits choline uptake into membrane material effectively (66 to 74%) under all medium conditions at 3 days (Table 2). At 10 or 19 days, however, this inhibition is much more effective in Hg medium (74% and 42% respectively) than in F or Fg media (55–66% and 6–17% respectively). Taken together with the results in Fig. 7, this evidence indicates that choline metabolism falls off more rapidly in F and Fg media than in Hg medium.

Table 2

Choline accumulation in membrane components of F, Fg and Hg cultures

Choline accumulation in membrane components of F, Fg and Hg cultures
Choline accumulation in membrane components of F, Fg and Hg cultures

Preliminary evidence from autoradiography of these choline-labelled cultures (data not shown) also tends to support this inference. Silver grains occur more frequently over neuronal aggregates than over flattened epithelial cells in the case of Hg but not F or Fg cultures, nor in Hg cultures incubated with 10 μM hemicholinium III. Owing to geometrical considerations, the grain densities over these different cell types cannot be compared directly, and the results obtained to date are merely indicative of higher neuronal uptake in Hg as compared to F or Fg media.

The results reported in this paper show that cholinergic neuronal differentiation in chick embryonic neuroretinal cultures is favoured by horse serum (in conjunction with high glucose), which also practically blocks later transdifferentiation into lens. The factors responsible for these effects have not been characterized as yet. Some inhibition of lens cell formation can be obtained with high glucose alone (i.e. in combination with foetal calf serum), while polylysine-coated substrates may promote neuronal differentiation without greatly affecting transdifferentiation. Several qualifications must therefore be made, if the main results are to be interpreted in terms of a choice between developmental pathways in culture.

First and foremost, neuroretinal cell cultures are not homogenous, but initially comprise a monolayer of flattened epithelial cells (glial Müller cells; Linser & Moscona, 1979), overlaid by aggregates of small rounded neuronal cells which become interconnected by long processes between the first and thirdweeks of culture. The neuronal identity of these aggreates has been demonstrated by staining with Merocyanine 540 (Okada et al. 1979), by tetanus toxin immunofluorescence (de Pomerai, preliminary results; Mirsky et al. 1978), and indirectly by the fact that cultures stripped of neuronal cells show negligible CAT activity compared with controls (this paper).

Secondly, there is as yet only indirect evidence that lens differentiation can be initiated within these neuronal aggregates as well as from the underlying epithelial cells (Okada et al. 1979). Intermediate structures apparently showing partial conversion of neuronal aggregates into lentoids are frequently encountered in cultures around the third week in vitro (de Pomerai & Clayton, 1980), and time-lapse film studies have shown cells within a neuronal aggregate swelling up and elongating into ‘bottle cells’ and lentoid structures during long term culture (K. Yasuda, pers. comm.; Okada et al. 1979). However, these data do not preclude the possibility that neuronal-glial cell interactions may promote lentoid development in vitro. The neurone-stripped cultures examined here retained a few neuronal aggregates ( < 10%), and of course large numbers were present up to the time of separation (day 4 in culture). To date there are no reported methods for obtaining cultures of completely purified epithelial or neuronal cell fractions from retinal material, in order to determine whether either alone can produce lentoids in vitro.

A third problem is that markers of cholinergic neuronal differentiation decline to low levels well before lentoids begin to appear in neuroretinal cultures. This is true whether the lentoids appear early and in large numbers (F medium) or late and very sparsely (Hg medium). Numerous neuronal aggregates and even some interconnecting processes can be found in 5-to 6-week-old neuroretinal cultures maintained in Hg or Fg media (Fig. 3), but these no longer possess high levels of cholinergic markers (Fig. 7). However, preliminary evidence suggests that they can still be stained by tetanus toxin immunofluorescence (de Pomerai, unpublished) and may thus remain ‘neuronal’ in terms of some cell surface features. This might imply gradual dedifferentiation of retinal neuronal cells under in vitro culture conditions, with cholinergic markers being lost more rapidly in some media (F and especially Fg) than in others (Hg).

It has been shown previously that crystallin production in F cultures first becomes detectable between 12 and 16 days in vitro (de Pomerai & Clayton, 1978; de Pomerai et al. 1977). During this phase of culture, cholinergic markers are at low levels in F and Fg media, but remain relatively high (albeit declining) in Hg medium. Further experiments are needed to establish the true importance of this phase, but ideally they will require the development of medium conditions able completely to abolish transdifferentiation into lens.

A final problem is the possibility that differential culture growth, cell density, and (most crucially) cell selection by the medium, might substantially affect these results. At least the first two of these parameters are known to affect the extent of transdifferentiation in chick neuroretinal cultures (Clayton, de Pomerai & Pritchard, 1977; de Pomerai & Clayton, 1980). However, the differences in culture growth rate and final cell density are only about twofold between Fg and Hg media and F medium (Fig. 1), yet the inhibition of crystallin production by Hg medium (Fig. 5 A) is far greater than that produced by Fg medium (Fig. 5 A) or by a 10-fold difference in the initial cell density under F conditions (Clayton et al. 1977). It might be argued that the greater cell density in Hg medium (as compared to F or Fg media) during the first 12 days of culture (see Fig. 1) could favour CAT activity (Fig. 7) via a non-specific celldensity effect. In fact, the data in Fig. 7 were derived from a number of separate culture experiments in which the initial cell densities varied from 3 to 7 × 106 cells per ml. Even in the low-density Hg cultures, CAT activity per μg protein remained consistently higher than in high-density F or Fg cultures. Thus, a non-specific cell density effect on CAT activity seems unlikely, just as the later differences in growth rate seem inadequate to account for the differential effects of F and Hg media on crystallin production.

The cell size distribution in Fig. 2 show that cultures in Fg medium are depleted in cells belonging to the larger (10–16 μm diameter) size-classes (Fig. 2, 10 and 20 days), whereas these cells are prominent in both F and Hg media. While this result does support the possibility of cell selection occurring in neuroretinal cultures, there is no obvious correlation with any of the differentiation parameters assessed here. There are suggestions that the largest class of cells in embryonic chick neuroretina may be predominantly ganglion cells, as shown by Percoll fractionation of 14-day chick embryo retina cells (Sheffield, Pressman & Lynch, 1980). Further work is in progress to characterize the various size classes of retinal cell by means of specific neuronal markers, and also Müller cell markers such as glutamine synthetase (Linser & Moscona, 1979) and glial fibrillary acidic protein (Bignami & Dahl, 1979).

The authors would like to thank Drs N. L. Robinson and I. R. Duce for advice on the assessment of neuronal differentiation, Mr R. Searcy for preparing the photographs, and Mrs R. Clayton (University of Edinburgh) for generous gifts of anticrystallin antisera. M.A.H.G. is in receipt of a postgraduate training award from the Government of the Republic of Iraq. This work was supported by a grant from the Medical Research Council.

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