The in vitro transdifferentiation of chicken embryo neural retina into pigment epithelium and lens cells was investigated under a variety of experimental conditions. Our findings suggest that some aspects of the phenomena are a function of medium composition and volume, whereas others depend upon conditions which develop during culture growth. Before melanin is visible, potential pigment cells are recognized as foci within epithelial sheets which remain in contact with the dish. The final area occupied by colonies of potential pigment cells is directly proportional to bicarbonate concentration. Low total medium volume also favours formation of potential pigment cells. In contrast the extent of cells other than potential pigment cells is not related to bicarbonate and is favoured when the volume of medium is large. Accumulation of melanin within the potential pigment cell colonies is suppressed when cells are crowded together. Lentoid bodies are formed from cells which are distinct from potential pigment cells and arise in crowded situations, in association with multilayering. Another type of structure superficially resembling a lentoid is derived from cell aggregates formed during the initial establishment of cultures. The survival of these ‘aggregate bodies’ is inversely related to bicarbonate concentration. Crystallin content is unrelated to lentoid numbers. The results provide the basis for a new hypothesis concerning cytodifferentiation in this system.

In certain biologically unusual situations, animal cells can lose their definitive characteristics and instead acquire those features which normally characterize alternative differentiated states. This phenomenon, termed ‘metaplasia’ or ‘transdifferentiation’, is of considerable significance both to oncologists and developmental biologists. Chicken embryo neural retina is of particular interest in this respect since this tissue, when dissociated and re-established in vitro, has the capacity to transdifferentiate into lens-like structures termed ‘lentoid bodies’, which contain high concentrations of the lens-specific crystallins (Okada, Itoh, Watanabe & Eguchi, 1975; de Pomerai, Pritchard & Clayton,1977), as well as into pigmented epithelium (Itoh, Okada, Ide & Eguchi, 1975), with dense deposits of melanin. This implies that either embryonic neural retina contains cells already partially differentiated towards other tissue types, or else that culture conditions can significantly alter gene expression in these cells.

In certain amphibian and mammalian systems, initiation of melanin synthesis is stimulated by specific ions (Barth & Barth, 1974a, b; Lerner, 1955). The transdifferentiation of neural retina is also affected by the choice of culture medium (Okada, 1976). We have carried out a detailed analysis of the development of lentoid bodies and pigment epithelium in several media and have examined the effects produced by varying the concentrations of specific medium constituents. We have also tested the effects of chilling, since in many vertebrates, deposition of melanin is enhanced at prolonged low temperatures (Fox & Vevers, 1960).

The experimental work falls into two parts. A suitable medium was first selected and detailed observations of the cultures were made by phase-contrast microscopy and time-lapse photography. The effects of sodium and potassium concentrations, genotype and chilling were also assessed in the preliminary experiments. In the second part a detailed comparative stereological analysis was made of cultures grown in three media with different capacities for supporting transdifferentiation.

Culture conditions

Cultures were established as described by Okada et al. (1975) from dissociated neural retina taken from 8- to 9-day chicken embryos of a control, brown feathered strain, N, with eyes of normal morphology and pigmentation, and a white feathered strain, Hy-1, which has been observed to exhibit abnormalities of lens (Clayton, 1975; Clayton et al. 1976, Eguchi, Clayton & Perry, 1975) and comparable abnormalities of the retina (Pritchard & Clayton, 1978) and in which pigment is confined to the tapetum and iris and is absent from the choroid. The cell preparations were examined by high and low power microscopy to ensure no possible contamination by cells of other types. Tissue culture media and supplements were supplied by Gibco Biocult Limited, Paisley, Scotland. All media were supplemented with 6% foetal calf serum, 100i.u./ml penicillin and 100μg/ml streptomycin. Medium was replenished every second day.

It has been shown that Minimal Essential Medium (‘MEM’; Eagle, 1959), permits transdifferentiation of neural retina cultures (Itoh et al. 1975; Okada, 1976; Okada et al. 1975). This medium is available in several formulations. An initial comparison was made between N strain cultures set up and maintained in MEM based on Earle’s (1943) salts (‘EMEM’) containing 2·20 g/1 sodium bicarbonate and in MEM based on a modified version of Hanks’ salts (Hanks & Wallace, 1949), in which the sodium bicarbonate content was increased from 0·35 g/1 to 1·40 g/1 (‘mod. HMEM’). In an atmosphere of 5% CO2:95% air, these two media equilibrate at the same pH. In later experiments unmodified Hanks’ medium (‘HMEM’) was also used, which attains the same pH in a restricted atmosphere of 100% air. The relative concentrations of sodium and potassium in EMEM were changed by completely replacing the sodium bicarbonate with an equimolar concentration of potassium bicarbonate (‘EMEMK’). The ionic concentrations of sodium, potassium and bicarbonate in the four media are shown in Table 1.

Table 1

Concentrations of sodium, potassium and bicarbonate ions in the media as formulated

Concentrations of sodium, potassium and bicarbonate ions in the media as formulated
Concentrations of sodium, potassium and bicarbonate ions in the media as formulated

Cultures were grown routinely in polystyrene Petri dishes of floor area 20 cm2 (Falcon ref. 3002), within sealed boxes filled with the appropriate gas phase. In the later experiments, stoppered polystyrene tissue culture bottles of floor area 25 cm2 (Falcon ref. 3012) filled with filtered gas were also used, while the Petri dishes were placed in a gassed incubator; in these conditions, the medium was maintained at about 0·3 pH units below that in the former situation. Dishes were inoculated at around 5 × 106 cells in 5 ml of medium and bottles at around 7 × 106 cells in 10 ml of medium.

Cultures were normally maintained at 37°C, but in the experiment to test the effects of chilling, they were transferred to a 29° incubator at the time when pigment was first being deposited. After 8 days, chilled cultures were returned to the 37° incubator and after a further 8 days, were assessed relative to controls maintained at 37° throughout.

Assessment of cultures

In the preliminary experiments the areas of the dishes occupied by pigmented cells were estimated stereologically using a regular lattice placed under the dish, or over a photographic enlargement (Elias, 1965). In later experiments dishes and bottles were examined at each of 16 sites, at the intersections of a hypothetical lattice of 1 cm intervals, defined by the reference scales on the stage of a Gillett and Sibert inverted, phase-contrast ‘Conference’ microscope. The occurrence of pigmented and potential pigment cells, multilayers, extracellular membrane and uncolonized vessel floor at each of the intersections was recorded, as well as the number of lentoids in the whole field visible at each intersection. The percentage area of the vessel floor occupied by each cell type and the number of lentoids per unit area of floor were calculated from these figures. The data was also plotted in the form of a topological frequency diagram for each feature. The collection of stereological data and time-lapse photography of areas of interest were facilitated by the apparatus of Pritchard & Ireland (1977).

Cell counts were made with a modified Fuchs-Rosenthal haemocytometer (BS 748) after trypsinization and resuspension of the cells.

Estimates of the relative amounts of crystallin subunits were made by optical scanning of stained polyacrylamide gels, after electrophoresis of the samples in the presence of sodium dodecyl sulphate and urea (MacGillivray, et al. 1972). Scanning was carried out with the Kipp and Zonen KS 3 densitometer.

(A) Selection of medium

Cultures grown in EMEM consistently developed more pigment than those in mod. HMEM (Figs. 1A, 1B, Table 2), about five times as many pigmented colonies were developed and these occupied ten times as large an area as those in mod. HMEM. EMEM was therefore selected for routine use. A growth curve of N-strain cells in EMEM is shown in Fig. 2.

Table 2

Pigmentation developed in mature cultures of neural retina grown in different media

Pigmentation developed in mature cultures of neural retina grown in different media
Pigmentation developed in mature cultures of neural retina grown in different media
Figure 1

(A, B) Unstained terminal cultures of neural retina photographed by transmitted light. (A) Culture established and grown in mod. HMEM and (B) in EMEM (see Table 1). (C-J) Neural retina cultures grown in EMEM photographed under phase-contrast illumination. The bar represents 100μm. (C) A developing colony of pigment epithelium. Note pigmented cells at top left and bare dish at lower right. (D) The same field as (C), 5 days later. (E) A mature colony of pigment epithelium with adjacent lentoids. (F) A lentoid in the upper sheet of the multilayer. (G) A large aggregate which appeared in a late culture. (H) Small, linked aggregates in an early culture. (J) Strand of extracellular material in an old culture.

Figure 1

(A, B) Unstained terminal cultures of neural retina photographed by transmitted light. (A) Culture established and grown in mod. HMEM and (B) in EMEM (see Table 1). (C-J) Neural retina cultures grown in EMEM photographed under phase-contrast illumination. The bar represents 100μm. (C) A developing colony of pigment epithelium. Note pigmented cells at top left and bare dish at lower right. (D) The same field as (C), 5 days later. (E) A mature colony of pigment epithelium with adjacent lentoids. (F) A lentoid in the upper sheet of the multilayer. (G) A large aggregate which appeared in a late culture. (H) Small, linked aggregates in an early culture. (J) Strand of extracellular material in an old culture.

Fig. 2

A typical growth curve of neural retina cells in EMEM (see Table 1). Each point represents the mean haemocytometer count of cells harvested from two dishes. (A) Foci of potential pigment cells present; crystallins detectable. (B) Multilayers present. (C) Melanin accumulates in potential pigment cells. (D) True lentoids present.

Fig. 2

A typical growth curve of neural retina cells in EMEM (see Table 1). Each point represents the mean haemocytometer count of cells harvested from two dishes. (A) Foci of potential pigment cells present; crystallins detectable. (B) Multilayers present. (C) Melanin accumulates in potential pigment cells. (D) True lentoids present.

(B) Development of pigmented colonies

The first deposits of melanin in cultures grown in EMEM are detectable at about 25 days. Pigmented cells are only found within colonies of similar, small, very closely packed cells, superficially resembling pigment epithelium from the tapetum (Cahn & Cahn, 1966; Eguchi & Okada, 1973; Itoh et al. 1975; Okada et al. 1975). These colonies invariably occur as monolayers, or, where a multilayer is present, in the lowest stratum, adjacent to the dish.

A pigmented colony arises in two recognizable phases. The first phase involves the establishment, at about 15 days, of a focus of some 50 small, unpigmented, polygonal ‘potential pigment cells’ within the sheet of more loosely packed cells (Fig. 1C). Each focus is surrounded by a ring of actively dividing cells and grows in area by incorporation of the daughter cells (Fig. 1D). About 10 days after the first signs of focal development, the central cells begin to accumulate pigment and pigmentation spreads outwards throughout the colony. Seven or eight days after the initiation of pigment synthesis, many of the older, pigmented cells become vacuolated and break down.

Figure 1C shows the focus of a small, pigmented colony among potential pigment cells, some of which are undergoing mitosis, compared with other monolayer cells shown in Fig. 1 J. Five days later the colony occupied a larger area (see Fig. 1C, D, lower right-hand corners) and many of the cells were pigmented. The minimum mean number of mitotic events which would account for the observed increase in cell density in the outer region of the colony is 1·4 per cell.

(C) Development of lentoid bodies and other features

From about 20 days, neural retina cultures consist of multilayered as well as monolayer expanses of tissue. As monolayer regions expand, heaps of cells appear around their perimeters, from which lentoids arise at about 33 days (Fig. 1E; cf. Okada et al. 1975). At the same time the multilayers contract laterally to form thicker masses and lentoids also arise from these (Fig. 1F). In addition, rounded eminences which resemble lentoids (‘aggregate bodies’) develop from small aggregates of cells (Fig. 1 H) which formed before adhesion of the inoculated cells to the substratum. Elongated cytoplasmic processes at first link these aggregates which were particularly numerous in mod. HMEM cultures, but virtually absent from those grown in EMEM (see below). In mod. HMEM cultures inoculated at 16 times the normal density, the survival of aggregates was remarkably high and the final combined total number of lentoids and aggregate bodies was quadrupled.

In the later stages, sheets and strands of extracellular material were seen overlying the cells (Fig. 1 J; cf. Redfern et al. 1976). Occasional large aggregates of cells were detected after about 30 days, particularly where sheets of extracellular material were evident (Fig. 1 G).

(D) Strain comparison

Comparative growth curves of N and Hy-1 cultures are published elsewhere (Pritchard & Clayton, 1978). Pigmentation in cultures of Hy-1 neural retina did not differ significantly from that in N-strain genetic controls (results not shown).

(E) Effects of chilling and variation in concentrations of Na+ and K+ ions

In chilled cultures grown in EMEM orEMEMK the total area of the pigment colonies was reduced to nearly 50% (see Table 3), in association with the observed retardation of growth at the lower temperature.

Table 3

Pigmentation developed in chilled and control cultures of neural retina and in cultures grown in medium of high potassium content

Pigmentation developed in chilled and control cultures of neural retina and in cultures grown in medium of high potassium content
Pigmentation developed in chilled and control cultures of neural retina and in cultures grown in medium of high potassium content

Cultures grown in EMEMK developed a similar degree of pigmentation to those grown in standard EMEM (Table 3).

(F) Comparison of cultures in different media

Terminal cultures were compared after establishment and growth in EMEM, HMEM and mod. HMEM. Throughout most of the culture period, medium pH remained within the range 6·8–6·9 in all vessels, the significant difference between the media being the concentration of bicarbonate (Table 1).

With the exception of the data on the establishment of cultures, the following results are all based on one large-scale experiment, but the major findings are supported by less detailed observations from many experiments with different batches of medium and foetal calf serum.

Primary cultures from the same preparation of N-strain cells were established in each of the three media, in both types of vessel and were examined in detail at 60 days. Within each medium and vessel category there was little variation between replicates and the variation between repeated stereological assessments of the same culture was in every case smaller than the variation between assessments of replicates. Haemocytometer cell counts showed cell numbers in terminal cultures were more a function of the medium than of the conditions which differed between bottles and dishes (Table 4).

Table 4

Initial and terminal haemocytometer cell counts normalized with respect to floor area, total numbers of lentoid-like bodies (i.e. aggregate bodies plus true lentoids), and approximate crystallin content relative to total protein, in cultures grown in the three different media

Initial and terminal haemocytometer cell counts normalized with respect to floor area, total numbers of lentoid-like bodies (i.e. aggregate bodies plus true lentoids), and approximate crystallin content relative to total protein, in cultures grown in the three different media
Initial and terminal haemocytometer cell counts normalized with respect to floor area, total numbers of lentoid-like bodies (i.e. aggregate bodies plus true lentoids), and approximate crystallin content relative to total protein, in cultures grown in the three different media

I Medium effects. The influence of bicarbonate

(a) Establishment of cultures

Freshly inoculated cells stick down mainly as aggregates which spread to colonize the vessel floor, the haemocytometer count at 2 days being taken to indicate the number of cells which initially adhere to the dish. Later counts indicate the difference between cell survival plus gain by mitosis, and cell loss. Until 12 days there is a progressive net loss of cells (Fig. 2).

The haemocytometer count of adherent cells both at 2 and 6 days was inversely proportional to the bicarbonate content of the medium (Fig. 3).

Fig. 3

Haemocytometer counts of adherent cells at 2 and 6 days, plotted against sodium bicarbonate concentration in the media. Cultures were set up in HMEM, mod. HMEM, and EMEM (see Table 1). Each point represents the mean haemocytometer count of cells harvested from two dishes. Ranges of variation are smaller than the symbols.

Fig. 3

Haemocytometer counts of adherent cells at 2 and 6 days, plotted against sodium bicarbonate concentration in the media. Cultures were set up in HMEM, mod. HMEM, and EMEM (see Table 1). Each point represents the mean haemocytometer count of cells harvested from two dishes. Ranges of variation are smaller than the symbols.

(b) Potential pigment cells

Figure 4A shows the relationship between the bicarbonate concentration of the media and the area covered by colonies of pigment cell type, expressed as percentage coverage of the vessel floor, the areas occupied by pigmented and potential pigment cells being combined.

Fig. 4

(A) The relationship between sodium bicarbonate concentration in the medium, and the (combined) percentage area of the vessel colonized by (pigment and) potential pigment cells. Cultures were grown in dishes (●— ●) and bottles (▪— ▪). Each point represents the mean of four assessments, the standard error of the mean is denoted by an error bar unless smaller than the symbol. (B) The relationship between sodium bicarbonate concentration in the medium and total percentage area occupied by cells other than pigment and potential pigment cells. These values include the area of the upper sheet of the multilayer. (C) The relationship between sodium bicarbonate concentration in the medium and the percentage area of the vessel covered by the sheet of extracellular membranous material.

Fig. 4

(A) The relationship between sodium bicarbonate concentration in the medium, and the (combined) percentage area of the vessel colonized by (pigment and) potential pigment cells. Cultures were grown in dishes (●— ●) and bottles (▪— ▪). Each point represents the mean of four assessments, the standard error of the mean is denoted by an error bar unless smaller than the symbol. (B) The relationship between sodium bicarbonate concentration in the medium and total percentage area occupied by cells other than pigment and potential pigment cells. These values include the area of the upper sheet of the multilayer. (C) The relationship between sodium bicarbonate concentration in the medium and the percentage area of the vessel covered by the sheet of extracellular membranous material.

The values lie on two straight lines, one for dishes and one for bottles. This pattern shows that the development of colonies of potential pigment cells is directly related to the sodium bicarbonate concentration of the medium, but that other relevant conditions are different in the two types of vessel. It will be noted that the area occupied by potential pigment cells is more extensive in dishes than in bottles. The major difference between bottle and dish cultures is the volume of medium per unit area of vessel floor (see (c) below).

(c) Other cells, including potential lens cells

Figure 4B shows the area occupied by cells other than potential pigment cells, in relation to sodium bicarbonate concentration. In this case some values exceed 100% due to multilayering. In contrast to Fig. 4 A, the relationship is non-linear and the bottle values are higher than those for dishes. The ratio between bottle and dish values is 1·60 (±0·05): 1·00, equivalent to the ratio of the volumes of medium per unit area of vessel floor (1·60: 1·00).

(d) Extracellular material

The area of the sheet of extracellular material was related to sodium bicarbonate concentration in a positive, but non-linear fashion (Fig. 4C). No consistent difference between bottles and dishes could be found, nor was the extent or distribution of the sheet obviously related to any cell type.

II Effects of cell density

(a) Pigmentation

The micro-stereological estimates of the frequency of cells containing visible pigment produced values too small for analysis. The degree of pigmentation or ‘pigment expression’ of the cultures was therefore calculated as the ratio of the frequency of pigmented colonies to the combined frequency of pigmented and potential pigment cells, i.e. ‘pigment expression’ = number of pigment colonies per cm2/percentage area colonized by pigment plus potential pigment cells.

Since no cells of pigment cell type were detected in two stereological scans of the HMEM dishes, only a minimum value could be derived for these cultures. No pigment was seen in mod. HMEM bottles although potential pigment cells were present, so these cultures have a pigment expression value of zero.

Pigment expression was unrelated to bicarbonate concentration or the type of culture vessel, but was inversely related to cell density as estimated from the haemocytometer count (results not shown) and the stereological scan (Fig. 5 A). It is concluded that high density cultures will not produce pigment even if potential pigment cells are present, while in low density cultures expression of pigmentation is inversely related to cell density.

Fig. 5

(A) The relationship between pigment expression (i.e. number of pigmented colonies per cm2/percentage area colonized by pigment plus potential pigment cells) and total pecentage area of vessel colonized. Symbols as in Fig. 4. The lowest cell density point represents the mean of eight assessments and indicatesa minimum value for pigment expression. (B) Extent of the multilayer plotted against percentage area of the vessel colonized (b = 0·534 ±0·110).

Fig. 5

(A) The relationship between pigment expression (i.e. number of pigmented colonies per cm2/percentage area colonized by pigment plus potential pigment cells) and total pecentage area of vessel colonized. Symbols as in Fig. 4. The lowest cell density point represents the mean of eight assessments and indicatesa minimum value for pigment expression. (B) Extent of the multilayer plotted against percentage area of the vessel colonized (b = 0·534 ±0·110).

(b) Multilayering

Figure 5B reveals the positive relationship between multilayering and cell density. Multilayering occurs when floor coverage exceeds about 40%.

(c) Lent old bodies

True lentoids arise only in cultures that have undergone a net increase in cell numbers (see Table 4 and Fig. 2). In HMEM there was no net growth and no evidence of multilayering or true lentoids, but aggregate bodies were particularly numerous. Aggregate bodies were rarely seen in EMEM cultures in which initial survival was poor, but there was a relatively large net increase in total cell numbers and true lentoids developed. In mod. HMEM, cell survival to 6 days was exactly intermediate between that in the other media and both types of bodies were seen. The total numbers of lentoid-like bodies in the mature cultures are shown in Table 4.

Aggregate bodies and true lentoids were not counted separately, but our general observations suggest that the frequency of true lentoids is positively related to the area of the dish colonized, and correlates with the extent of the multilayer.

(d) Crystallin content of cultures

Alpha and β crystallins together contributed about 15% of the total protein in terminal EMEM and mod. HMEM cultures. In control preparations from day-old chick lens epithelium and whole lenses the contribution amounted to about 60% and about 30% respectively. (Separation of δ crystallin from retinal proteins of similar size was too poor for quantitation.) Although the total numbers of lentoid-like bodies (mainly aggregate bodies) in HMEM were much higher than in EMEM cultures the former samples produced only faint α and β crystallin bands representing a total of about 5% of the total protein (see Table 4).

The relative concentrations of α crystallin and the 23 000, 24 000–25 000 and 28000 dalton subunits of β crystallin in the assayable samples and, for comparison, in whole lenses and lens epithelium of day-old chickens, are shown in Fig. 6.

Fig. 6

Ratio of crystallins in mature neural retina cultures grown under different conditions, and in whole lenses and lens epithelium of day-old chickens. Estimates are based on densitometer scans of stained sodium dodecyl sulphate – urea – poly-acrylamide gels, (a) α crystallin; (b) 23000 dalton subunits of β crystallin; (c) 24000–25000 dalton subunits of β crystallin; (d) 28000 dalton subunits of β crystallin.

Fig. 6

Ratio of crystallins in mature neural retina cultures grown under different conditions, and in whole lenses and lens epithelium of day-old chickens. Estimates are based on densitometer scans of stained sodium dodecyl sulphate – urea – poly-acrylamide gels, (a) α crystallin; (b) 23000 dalton subunits of β crystallin; (c) 24000–25000 dalton subunits of β crystallin; (d) 28000 dalton subunits of β crystallin.

Cell culture experiments have revealed that, in contrast to the observations of Cahn & Cahn (1966), the normal restrictions upon the differentiated states of vertebrate eye tissues are not necessarily maintained when they are dissociated into single cells and allowed to grow in isolation from their normal neighbours (Eguchi & Okada, 1973). It is already well established that after extensive proliferation in cell culture, neural retina from chicken embryos can undergo changes which result in production of lens cells and pigment epithelium (Eguchi, 1976; Itoh et al. 1975; Okada, 1976, 1977; Okada et al. 1975). Our results confirm these conclusions and indicate that the conditions which facilitate development of the two cell types are distinct in several respects. High concentrations of sodium bicarbonate encourage growth of potential pigment cells, but facilitate loss of cell aggregates which would otherwise give rise to aggregate bodies. Small volumes of medium encourage pigment colony formation, whereas the spread of potential lens and other cells is encouraged by large volumes of medium. In cultures established at high inoculum densities lentoid-like bodies (including aggregate bodies) are abundant, whereas in primary cultures established at low inoculum densities, pigmented colonies are formed, but no lentoid bodies (Clayton, de Pomerai & Pritchard, 1977). Crowding in late cultures favours formation of true lentoids, but inhibits pigmentation.

Lentoids develop from the upper regions of multilayers, whereas pigment epithelium forms only among cells which are in contact with the vessel surface.

Differentiation of lens cells

De Pomerai et al. (1977) examined developing neural retina cultures by immunofluorescent microscopy and detected traces of crystallins in cell aggregates in freshly established cultures. These cultures were grown in EMEM and the majority of the aggregates were lost long before true lentoids developed. The crystallin composition of aggregate bodies per se has not been examined, but extracts of HMEM cultures, with many aggregate bodies, but few if any true lentoids and no multilayers, contained negligible levels of αor β crystallin (Table 4). Aggregate bodies therefore do not appear to be equivalent to true lentoids or lens cells.

Crystallins are again detectable by very sensitive haemagglutination techniques at 12–16 days (de Pomerai et al. 1977), 2 weeks or more before the appearance of true lentoids, whereas multilayering is visibly detectable at 20 days. After about 25 days, small areas of the cell sheet in EMEM dishes weakly bind fluorescent anti-crystallin antisera, but strongly immunoflurescent lentoids do not appear until 30 days (de Pomerai et al. 1977). The first steps along the differentiative pathway which ends in lens cells must therefore be taken long before true lentoids arise, but possibly not before the beginning of multilayering.

True lentoids tend to form in multilayers or where the margins of adjacent pigment cell colonies meet and the intervening cells pile up into ridges (Okada, et al. 1975). Lentoids arise 5 days earlier in cell sheets which have been folded (Clayton et al. 1977). Our general observations also suggest a correlation between numbers of true lentoids and extent of the multilayer. Lentoid growth therefore seems to be closely related to multilayering and crowding. In expiants of the lens epithelium lateral compression favours its differentiation into fibres (McLean & Finnegan, 1974).

Differentiation of pigment cells

The melanin granules which develop in neural retina cultures are of a different shape from those in the tapetum (Itoh et al. 1975), which suggests that the cells which contain them are not equivalent. In normal eyes the choroid also contains melanotic cells, the pigmentation of which is regulated independently of that in the tapetum (Weston, 1970), although choroidal and tapetal pigment cells have a common origin in the optic vesicle (Bartelmez & Blount, 1954; Coulombre, 1965). In Hy-1 birds the choroid does not become pigmented, a situation superficially comparable to that of black-eyed, white mice (Wolfe & Coleman, 1966). The strain comparison experiment was intended to test the possibility that the pigment epithelium which develops in neural retina cultures is derived from presumptive choroid cells which remained trapped in the presumptive neural retina (Okada et al. 1975). Negative results were obtained, but it is possible that the restrictions upon melanin synthesis in Hy-1 presumptive choroid cells would not be maintained in vitro (Whittaker, 1974). The possibility that neural retina preparations contain presumptive choroid pigment cells therefore remains unresolved.

During embryonic development of the chick eye, the tapetum differentiates from the neural retina from about the fourth day (Alexander, 1937; Coulombre, 1965; Coulombre & Coulombre, 1965; Dorris, 1938). Our material was from 8- to 9-day embryos at which stage some neural retina cells are still mitotically active (Coulombre, 1965; Fujita & Horii, 1963). Formation of pigmented tissue could therefore reveal a normal aspect of the potency of undifferentiated retinal cells. For normal differentiation of tapetum from the eye rudiment the cells in the outer optic cup must proliferate in a monolayer sheet which is under physical tension, and then cease proliferation (Lopashov, 1963; Coulombre, 1965). In our cultures also pigment cells differentiate only in colonies anchored directly to the dish, which have undergone proliferation and then ceased proliferating. Our observations therefore suggest that differentiation of pigment epithelium in these cultures proceeds in accordance with the critical conditions required by the presumptive tapetum in vivo. Crowding inhibits mitosis and pigmentation in neural retina cultures, but in our experience of cultures of tapetum from 8- to 9-day chicken embryos, crowding has no obvious inhibitory effect. Crowding therefore seems to affect the cytodifferentiation of pigment epithelium rather than melanin synthesis per se.

Okada (1976, 1977) has presented evidence that individual 8-day embryonic neural retina cells can produce either pigmented clones or lentoids, but not both. These cells, however, had been in culture for a considerable period before testing. Alternatively, neural retina cell populations might differentiate along alternative pathways in response to particular culture conditions. The implications of these two interpretations are significant. According to the first model the embryonic neural retina contains cells which already have a potential for differentiation towards foreign tissue types. The second model implies that major changes in gene expression can be brought about by very simple effectors. We suggest that culture conditions could initiate cytodifferentiation by selective utilization of alternative biochemical pathways concerned with growth, and that this is followed by selective amplification of low level syntheses characteristic of other cell types (see Clayton, 1978 a, b). It is possible, however, that culture conditions might also affect the course of differentiation by selectively favouring the growth of particular cell types present in the neural retina population (discussed in Clayton et al. 1977).

We have shown that the total area of the potential pigment cell sheet in neural retina cultures is directly related to sodium bicarbonate concentration (over the range considered), whereas that occupied by other cell types is not. Gross variation in the ratio of sodium to potassium had no obvious effect on the cultures, so it is inferred that the growth of foci of potential pigment cells probably involves processes which are promoted or limited by bicarbonate. We suggest that growth (i.e. increase in size and/or number) of neural retina cells, which results in potential pigment cells, occurs in conjunction with the utilization of biochemical pathways which operate only at high concentrations of bicarbonate, whereas growth of neural retina cells at low bicarbonate concentrations occurs with the preferential operation of a distinctly different set of biochemical pathways, which preclude development of the pigment cell phenotype. It is well known that the nutritional requirements for growth vary between different cell types (Holley, 1975; Paul, 1973), although the development and significance of such differences has apparently not been described in detail.

The actual accomplishment of mitosis seems to be an essential feature in the complete differentiation of pigment cells, as melanin is not accumulated in situations in which the mitosis of potential pigment cells is inhibited, apparently by crowding. It is noteworthy that several rounds of mitosis precede cytodifferentiative changes not only in cell cultures of eye tissues, but also in vivo, in other differentiating vertebrate systems (Gurdon & Woodland, 1970; Holtzer et al. and during ‘transdetermination’ of Drosophila imaginal discs (Hadorn, 1966). In Wolffian lens regeneration four complete cell cycles are required before the descendants of an iris epithelial cell become definitely dedifferentiated and a further two before they become irreversibly committed to metaplasia into lens cells (Yamada, 1976).

It has been shown in this laboratory that freshly excised neural retina from 8-day embryo chickens contains low concentrations of the most abundant mRNA species of the lens (presumably those coding for crystallins) (Jackson, et al. (1978)), whereas mature neural retina cultures have high levels of mRNA specific for α, β and δ crystallins (Thomson et al. 1978). Synthesis of melanin in neural retina cultures coincides with an increase in tyrosinase activity (Itoh et al. 1975). Thus transdifferentiation must involve major changes in the pattern of gene expression (at the level of protein and/or RNA synthesis), unless one supposes that a small subpopulation of partially differentiated lens or pigment cells is present in freshly excised neural retina, and can overgrow all other cell types under appropriate culture conditions.

As yet we have no convincing evidence whether the redirection of embryonic neural retina into new tissue types requires the selective multiplication of cells which, in vivo, are markedly different from one another in a manner which influences their ultimate fate; or whether the development of the cells is determined completely by the culture conditions (see also Clayton et al. 1977). It is hoped that this work will lead to experiments which will distinguish between these alternatives.

We are grateful to Mr J. Archibald and Mr A. Gristwood of Ross Poultry Limited and Dr T. C. Carter of the Poultry Research Centre who continue to supply us with fertile eggs and day-old chicks of various strains. We wish to thank Dr G. Bacon, Dr J. C. Campbell, Mr J. Cuthbert, Mr J. Jackson, Mr P. G. Odeigah, Dr R. C. Roberts, Dr I. Thomson, Dr D. E. S. Truman and Dr A. Wright for useful discussion and criticism. We are also grateful to Mrs C. Smart, Mrs M. McEwan and Mrs H. J. MacKenzie for technical help, Mr D. Chalmers and Mr A. McEwan for photographic work and Mr E. D. Roberts for drawing the figures. Our work is supported by the Cancer Research Campaign and the Medical Research Council. D. I. de Pomerai is supported by an M.R.C. Postdoctoral Training Fellowship.

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