Neural retina from 8- to 9-day embryo chickens was grown in long-term cell culture in an experiment to test the hpothesis that one step during the in vitro transdifferentiation of neural retina into pigment cells occurs in response to stimulation of tricarboxylic acid (TCA) cycle activity. Time-lapse photography showed that pigment-cell formation occurs through the intermediate stages of ‘undistinguished cells’, ‘pavement epithelium’ and ‘potential pigment cells’. Mitosis of undistinguished cells to pavement epithelium was proportional to malonate over most of the tested range of concentrations and was inhibited by succinate, which respectively depress and stimulate the TCA cycle. Conversely mitosis of pavement epithelium to potential pigment cells occurred in proportion to succinate concentration over most of the tested range and was inhibited by malonate, in support of the hypothesis under test.

Melanin synthesis begins in a minority of ‘pigment leader cells’ uniquely stimulated by the lowest concentration of malonate, although higher concentrations blocked pigment synthesis in all cell types. The pigment leader cells appear to act as centres of influence upon neighbouring potential pigment cells, which subsequently also beome pigmented. Lactate inhibited most or all of the steps in formation of pigment epithelium.

Between three and five mitoses occur in the production of pigment cells, whereas multilayers and lentoid bodies seem to be formed by expansion of undistinguished cells, probably without mitosis.

The observations lead to a general theory that metaplastic conversion between cell types in eye tissues may require the physical isolation of overtly differentiated, multipotent cells from ‘leader’ cells which normally hold them in physiological subjugation.

The in vitro ‘transdifferentiation’ of 8- to 9-day chicken embryo neural retina (NR) into lens fibre-like cells and pigment epithelium, first reported by T. S. Okada’s group at Kyoto (Okada, Itoh, Watanabe & Eguchi, 1975; Itoh, Okada, Ide & Eguchi, 1975), is now a well-accepted phenomenon (see reviews by Clayton, 1978, 1979 and Okada, 1976). The direction and degree of change can be controlled by modification or choice of culture medium (Clayton, de Pomerai & Pritchard, 1977; Itoh, 1976; Okada, 1976, 1977; Pritchard, Clayton & de Pomerai, 1978); by selection of embryos of particular ages (Araki & Okada, 1978; Nomura & Okada, 1979; de Pomerai & Clayton, 1978); by adjusting the cell inoculum density; and by formation of artificial multilayers (Clayton et al. 1977). Significantly, the area occupied by the immediate pigment-cell precursors, or ‘potential pigment cells’, is directly proportional to bicar-bonate over the range covered by standard media, whereas that occupied by other cell types is not (Pritchard et al. 1978).

Retinal tissues show significantly different physiological behaviour in bi-carbonate-buffered, as compared with phosphate-buffered medium (Graymore, 1969) due to incorporation of bicarbonate ions into the tricarboxylic acid (TCA) cycle (Crane & Ball, 1951a, b). It was to test the hypothesis that TCA-cycle stimulation through CO2 fixation was the basis of the bicarbonate effect upon transdifferentiation, that the experiments described below were carried out.

Cultures of chicken embryo NR were treated with sodium succinate and sodium malonate respectively to stimulate and inhibit the TCA cycle (Lehninger, 1972). One expected outcome of TCA-cycle inhibition would be the increased conversion of pyruvate into lactate. To control for the effects of lactate, as distinct from TCA-cycle inhibition per se, cultures were grown also in medium supplemented with sodium lactate. The results support the hypothesis that control of TCA-cycle activity is a primary feature of the transdifferentiation of neural retina into pigment cells.

Neural retina cultures were established from trypsin-dissociated tissues taken from 8-to 9-day chicken embryos, as described by Okada et al. (1975). They were grown in loosely stoppered polystyrene tissue-culture flasks, in modified or standard versions of Minimal Essential Medium (Eagle, 1959) based on Earle’s salts, ‘EMEM’ (Earle, 1943), supplemented with 6% foetal calf serum which was taken from a single batch, 100i.u./ml penicillin and 100 μg/ml streptomycin (all materials from Gibco Europe). The cells were inoculated at 7 × 106 per flask of floor area 25 cm2 and grown at 37°C in a humid atmosphere of 5% CO2 in air,

In the main experiment 40 identical, serially numbered cultures were set up, each in 5 ml of standard EMEM, which was replaced with 8 ml on day 2. On day 6 they were divided into sets of four, each set containing numbers distributed throughout the series. From this time onwards one control set received 8 ml of standard EMEM, while the other sets all received 8 ml of EMEM supplemented with 1·5,0·75 or 0·15 g/1 of the appropriate biochemical: disodium succinate hexahydrate (Sigma) at 5·6 (SH), 2·8 (SI) or 0·56 mM (SL); disodium malonate monohydrate (Sigma) at 8·9 (MH), 4·5 (MI) or 0·9 mM (ML) and sodium lactate (L(+) lactic acid (Sigma grade L-l) titrated to neutrality with sodium hydroxide) at 16·7 (LH), 8·4 (LI) or 1·7 HIM (LL). This supplementation did not affect the pH of the media.

The medium was replenished every two or three days and the cultures were assessed stereologically with a phase-contrast microscope around day 60, at 30–100 sites per flask.

Six further cultures set up from the same cell preparation were given standard EMEM up to day 56. They were then divided into two equally pigmented sets of three. One set was given medium supplemented with 16·7 HIM sodium lactate, while the others continued receiving standard EMEM. These Tate lactate’ cultures and their controls were assessed and harvested on day 76.

Four cultures grown under control conditions were examined by time-lapse photography at two fields per culture. Relocation of the fields was achieved by the method of Pritchard & Ireland (1977).

The growth curve was based on haemocytometer counts of suspended cells from three replicate cultures at each time point.

Aggregate bodies

During the earliest stages of culture development, the ‘minute cells’ of the freshly dissociated NR settle down singly, or in ‘aggregate bodies’ (Pritchard et al. 1978) composed of many cells, before expanding and spreading over the vessel floor as a sheet of large ‘undistinguished cells’ with no major distinctive features (see Fig. 1B,C). At day 21, cultures supplemented with succinate, LL and LI contained normal numbers of aggregate bodies, but LH and all concentrations of malonate produced marked reductions.

Fig. 1

(A) Large and minute cells (some arrowed) in a suspension prepared by trypsinization of an established culture. (B-E) The same area of a control culture grown in standard EMEM and photographed under phase-contrast illumination at different stages of development. (B) Aggregate bodies at day 3. (C) Undistinguished cells with two aggregate bodies at day 14. (D) Pavement epithelium at day 37. (E) Potential pigment cells at day 53. (F, G) The same area of a culture at day 53 photographed by normal, direct light (F) and phase-contrast illumination (G), showing melanin in the pigment leader cells. All photographs are to the same scale, the bar represents 100 μm.

Fig. 1

(A) Large and minute cells (some arrowed) in a suspension prepared by trypsinization of an established culture. (B-E) The same area of a control culture grown in standard EMEM and photographed under phase-contrast illumination at different stages of development. (B) Aggregate bodies at day 3. (C) Undistinguished cells with two aggregate bodies at day 14. (D) Pavement epithelium at day 37. (E) Potential pigment cells at day 53. (F, G) The same area of a culture at day 53 photographed by normal, direct light (F) and phase-contrast illumination (G), showing melanin in the pigment leader cells. All photographs are to the same scale, the bar represents 100 μm.

Minute and large cells

When released by trypsinization, the undistinguished cells and their derivatives adopt a spherical shape of diameter 10–20μm, whereas those which have not expanded become spheres of 2–5 μm diameter (Fig. 1A). Minute cells multiply at a rate similar to that of the large cells (Fig. 2) although the origin of new minute cells is not known.

Fig. 2

Growth curves of large, minute and total neural retina cells in a control set of cultures grown in standard ENEM. Each point represents the mean haemo-cytometer count of cells harvested from three flasks. ●, Total; ○, large; ●, minute.

Fig. 2

Growth curves of large, minute and total neural retina cells in a control set of cultures grown in standard ENEM. Each point represents the mean haemo-cytometer count of cells harvested from three flasks. ●, Total; ○, large; ●, minute.

Large cell numbers showed a similar pattern of response to the supplements to that for production of potential pigment cells (Fig. 6B), but minute cell numbers were less variable.

Total area of cell sheets

The final total areas of the cell sheets are shown in Fig. 3. Lactate and malonate depressed colonization of the vessel, while succinate marginally promoted it. Malonate caused considerable death of undistinguished cells.

Fig. 3

The relationship between percentage areas of vessels occupied by cells and the concentrations of supplements in the medium. Cultures were assessed around day 60. The supplements were disodium succinate hexahydrate, disodium malonate monohydrate and lactic acid titrated to neutrality with sodium hydroxide. Each point represents the mean assessment of four cultures, the standard error of the mean is denoted by an error bar unless smaller than the symbol., – –succinate; – – –lactate;, — malonate.

Fig. 3

The relationship between percentage areas of vessels occupied by cells and the concentrations of supplements in the medium. Cultures were assessed around day 60. The supplements were disodium succinate hexahydrate, disodium malonate monohydrate and lactic acid titrated to neutrality with sodium hydroxide. Each point represents the mean assessment of four cultures, the standard error of the mean is denoted by an error bar unless smaller than the symbol., – –succinate; – – –lactate;, — malonate.

Gross appearance of mature cultures

The gross appearance of typical terminal cultures is shown in Fig. 4. Cultures grown in MI, MH and LH conditions developed no pigment colonies. Under LI and succinate supplementation their numbers were greatly reduced, but ML and LL conditions produced no marked effect (see Table 1).

Table 1

The effects of the supplements upon pigmentation

The effects of the supplements upon pigmentation
The effects of the supplements upon pigmentation
Fig. 4

Typical neural retina cultures photographed at day 60, showing variation in pigmentation. (A) Control culture grown in standard EMEM. (B-K) Cultures grown in medium supplemented with low, intermediate and high levels of succinate (B, C, D), malonate (E, F, G) and lactate (H, J, K).

Fig. 4

Typical neural retina cultures photographed at day 60, showing variation in pigmentation. (A) Control culture grown in standard EMEM. (B-K) Cultures grown in medium supplemented with low, intermediate and high levels of succinate (B, C, D), malonate (E, F, G) and lactate (H, J, K).

Sequence of differentiative events

Time-lapse photographic analysis of cultures raised in standard EMEM revealed the sequence of morphological changes which occur as undistin-guished cells differentiate into pigment epithelium (Figs. 1B-E, 8). The same sequence was recorded in three fields and accords with my general observations of several hundred similar cultures.

From days 12–16 some of the undistinguished cells, of in situ diameter about 37 μm, divide once or twice and their outlines become more discrete as they form a pavement epithelium with cells of mean in situ diameter about 26 μm. These cells then subdivide a further two or three times to produce potential pigment cells of about 12 μm diameter (Pritchard et al. 1978), which accumulate pigment from about day 40. In a minority of areas the intermediate pavement epithelium stage is not detectable. The process of subdivision of pavement cells spreads outwards from scattered foci (Pritchard et al. 1978).

Proportion of cell types in mature cultures

The relative areas occupied by the different cell types in mature cultures are shown in Fig. 5. There were great variations in their proportions.

Fig. 5

Diagram to show relative percentage areas of vessels occupied by undistinguished cells (▫), pavement epithelium (▦) potential pigment (▨) and pigment cells (▪) in terminal cultures of neural retina. (A-K) as in Fig. 4.

Fig. 5

Diagram to show relative percentage areas of vessels occupied by undistinguished cells (▫), pavement epithelium (▦) potential pigment (▨) and pigment cells (▪) in terminal cultures of neural retina. (A-K) as in Fig. 4.

Fig. 6

(A) The relationship between the percentage area of the cell sheet which becomes converted into pavement epithelium, or remains as undistinguished cells, and concentrations of supplements in the medium. (B) The relationship between the percentage area of pavement epithelium which becomes converted into potential pigment cells, and concentrations of supplements in the medium. Details as in Fig. 3.

Fig. 6

(A) The relationship between the percentage area of the cell sheet which becomes converted into pavement epithelium, or remains as undistinguished cells, and concentrations of supplements in the medium. (B) The relationship between the percentage area of pavement epithelium which becomes converted into potential pigment cells, and concentrations of supplements in the medium. Details as in Fig. 3.

Transdifferentiation was blocked before the potential-pigment-cell stage in LH cultures and at the melanogenic stages by MI and MH conditions.

Formation of pavement epithelium

Figure 6 A shows the fractions of the cell sheets which became converted into pavement epithelium, these values being derived by combining the areas finally occupied by pavement, potential pigment and pigment cells. Formation of pavement epithelium was proportional to malonate concentration, although only MH caused an increase relative to the control. Both lactate and succinate produced significant reductions.

Formation of potential pigment cells

The proportions of pavement epithelium which became subdivided into potential pigment cells are shown in Fig. 6B. Malonate and lactate strongly inhibited this step, but potential-pigment-cell formation was proportional to succinate concentration, although no treatment caused an increase relative to the control. Since this is the major mitotic step, this pattern was closely similar to that of large cell numbers (results not shown).

Development of pigmentation

Melanogenesis develops only in large spreads of potential pigment cells and begins in the foci where potential pigment cells first appeared. Groups of about twenty ‘pigment leader cells’ begin to synthesize pigment with no change of form (Pritchard et al. 1978), or three or four adjacent cells expand, forcing their neighbours to adopt a concentric pattern about them, before they become pigmented (Fig. 1F, G). Pigmentation then spreads outwards from these foci.

The relative frequencies of sites of initiation of pigmentation were estimated by relating numbers of pigment colonies to areas of cells which reach the potential-pigment-cell state (see Table 1). Initiation of pigmentation was unaffected by succinate or lactate. It was completely inhibited in MI and MH cultures, but interestingly, ML conditions caused a significant in crease.

Efficiency of spread of melanogenesis through the potential pigment cells is indicated by the size of the pigment colonies (Table 1). Mean colony area was significantly reduced by lactate and SH, but not SL or SI. The effects of MI and MH were not tested, as no colonies were initiated, but significantly ML had no effect, suggesting that spread of pigment synthetic activity is biochemically distinct from that of its initiation.

The ‘late lactate’ experiment

The observations recorded in Table 2 suggest that lactate probably exerts an inhibiting effect at most stages in the production of pigment cells, but this is statistically significant only at the conversion of pavement epithelium to potential pigment cells.

Table 2

The effect of lactate on mature cultures

The effect of lactate on mature cultures
The effect of lactate on mature cultures

Undistinguished cells and multilayers

The proportion of undistinguished cells was markedly increased in all experimental cultures, except MI and MH, in which there was much cell death (Figs 5, 6A). Comparison of Figs 6A and 7 demonstrates that the areas occupied by undistinguished cells and multilayers are affected by the supplements in similar ways, but to different degrees, in accordance with the theory that undistinguished cells produce multilayers by upward extrusion of cells. There is no indication that undistinguished cells are produced by any means other than expansion of minute cells, and detailed counts of the different cell types suggest that in these mass cultures (cf. clonal cultures, Okada et al. 1979) undistinguished cells do not undergo mitosis without yielding pavement epithelium.

Fig. 7

The relationship between the percentage area of the cell sheet which becomes multilayered, and concentrations of supplements in the medium. Details as in Fig. 3.

Fig. 7

The relationship between the percentage area of the cell sheet which becomes multilayered, and concentrations of supplements in the medium. Details as in Fig. 3.

Figure 8 summarizes a tentative interpretation of the sequence of differentiative changes in long-term NR cultures and the biochemical influences which affect them. One or more of the supplements exerted a significant effect on every variable examined, and it is concluded that the TCA cycle has a profound effect on the transdifferentiation of NR cells.

Fig. 8

Tentative scheme of differentiation in long-term mass cultures of neural retina from 8- to 9-day chicken embryos. Minute cells (I), in young cultures expand to form undistinguished cells (II, III) which produce multilayers (IV) probably by extrusion, and then lentoid bodies (V). Undistinguished cells divide to form pavement epithelium (VI), which on further mitosis yields potential pigment cells (VII, VIII). The latter occasionally arise direct from undistinguished cells. Pigment synthesis occurs first at pigmentation initiation foci (VIII) containing pigment leader cells, then spreads outwards to produce pigment epithelium (IX). Occasionally lentoid bodies arise within the pigment epithelium. Step I-II is inhibited by bicarbonate (Pritchard et al. 1978) and steps II-1V by malonate. Step II-VI occurs in the presence of malonate, but is inhibited by succinate and lactate. Step VI-VII is promoted by bicarbonate (Pritchard et al. 1978) and occurs in the presence of succinate, but is inhibited by lactate and malonate. Step VII-VIII is promoted by low concentrations of malonate, but inhibited by lactate and intermediate and high concentrations of malonate. Step VIII-IX is inhibited by lactate. M, Observed mitotic events.

Fig. 8

Tentative scheme of differentiation in long-term mass cultures of neural retina from 8- to 9-day chicken embryos. Minute cells (I), in young cultures expand to form undistinguished cells (II, III) which produce multilayers (IV) probably by extrusion, and then lentoid bodies (V). Undistinguished cells divide to form pavement epithelium (VI), which on further mitosis yields potential pigment cells (VII, VIII). The latter occasionally arise direct from undistinguished cells. Pigment synthesis occurs first at pigmentation initiation foci (VIII) containing pigment leader cells, then spreads outwards to produce pigment epithelium (IX). Occasionally lentoid bodies arise within the pigment epithelium. Step I-II is inhibited by bicarbonate (Pritchard et al. 1978) and steps II-1V by malonate. Step II-VI occurs in the presence of malonate, but is inhibited by succinate and lactate. Step VI-VII is promoted by bicarbonate (Pritchard et al. 1978) and occurs in the presence of succinate, but is inhibited by lactate and malonate. Step VII-VIII is promoted by low concentrations of malonate, but inhibited by lactate and intermediate and high concentrations of malonate. Step VIII-IX is inhibited by lactate. M, Observed mitotic events.

The production of potential pigment cells from pavement epithelium occurred in proportion to succinate concentration and was inhibited by malonate, in support of the hypothesis under test, namely that the promotory effect of bicarbonate upon production of potential pigment cells involves TCA-cycle stimulation through CO2 fixation (see Introduction). However, relative to the unsupplemented control, this involved depression of mitosis at SL and no increase, even at SH (Fig. 6B). Possibly the TCA cycle cannot be stimulated to greater activity than that in EMEM, which contains a very high concentration of bicarbonate, but the depression of mitosis by SL requires explanation. There was a similar depression in the mitosis of pavement epithelium to potential pigment cells due to ML, despite its increase with malonate concentration (Fig. 6A). Also initiation of pigmentation was significantly increased compared with controls, by ML, but MI and MH completely inhibited it. Three possible explanations are offered for these non-linear effects:

(i) Some differentiative events occur only, or optimally, at critical levels of TCA-cycle activity.

(ii) Important subsidiary biochemical pathways involving the supplements may be more susceptible to low-level supplementation than the TCA cycle itself.

(iii) The supplements may evoke compensatory responses which over-com-pensate at low levels of supplementation.

Low concentrations of malonate significantly boosted initiation of pigment synthesis in the pigment leader cells, but had no effect on its subsequent spread through the potential pigment cells. This implies that the leaders are particularly responsive cells which, using a different biochemical means, influence their less responsive neighbours to adopt, overtly at least, a similar phenotype. Pigment density tends to be uniform within any one colony, although variable between colonies in the same culture, suggesting physiological communication between contiguous cells (see Loewenstein, 1968). The concept that multipotent follower cells are only overtly subjugated by the physiology of neigh-bouring determined leaders might explain the capacity of pigment epithelial cells from mature NR cultures to transdifferentiate into lens when isolated in subculture (Okada, Yasuda, Araki & Eguchi, 1979). A variety of other phenomena of regeneration, metaplasia and transdifferentiation of eye tissues could also be explained if the same situation applies in vivo (see reviews by Clayton, 1978, 1979; Coulombre, 1965; Eguchi, 1979; Lopashov, 1963; Okada, 1976; Yamada, 1976.

In Wolffian lens regeneration in lentectomized newt eyes, Yamada (1976) reported that six cell divisions are required for the irreversible metaplasia of pigment epithelium into lens. In mass cultures of NR from 8- to 9-day chicken embryos, very little or no mitosis seems to occur in the differentiation of lens cells (although the evidence is indirect, being based on counts of other cell types), but crowding does seem to be essential (see Pritchard et al. 1978). In contrast, during pigment-cell formation, cell counts indicate that between three and five mitoses occur (see Fig. 1C-E). Of these the first one or two require an inactive TCA cycle, whereas the remainder require TCA-cycle activity. Mitosis would seen necessary for reduction of cell volume, but as yet there is no evidence that it is a prerequisite for melanin synthesis. Cessation of mitosis probably is essential for melanin synthesis (Whittaker, 1974).

The most significant outcome of this work is the discovery that imposition of a defined, but atypical physiological regime upon a vertebrate cell type can cause it to change to a recognizably different one. Other authors have shown that such changes involve modifications in the patterns of gene expression (Itoh et al., 1975; Okada et al. 1975; de Pomerai, Pritchard & Clayton, 1977; Thomson, de Pomerai, Jackson & Clayton, 1979), possibly by amplifying low-level syntheses of mRNAs characteristic of the new cell types (Clayton, 1978, 1979; Jackson et al. 1978). Examples of the control of gene expression by simple physiological stimuli are rare in eukaryotes. The identification in this paper of an area of biochemistry significant in this system, opens the way for a detailed analysis of the intra- and extracellular biochemical conditions which initiate and stabilize new patterns of gene activity during differentiation of eukaryote cells. Work in progress in this laboratory is directed toward this goal.

It is with pleasure that 1 acknowledge the encouraging interest in this work shown by my friends, relatives and colleagues. In particular I wish to thank Professor D. F. Roberts, Dr S. S. Papiha, and my wife Penny, for reading and criticizing the manuscript. Thanks are also due to Mr M. Booth and Miss F. Behjati for technical assistance, and to Mrs P. Dun-woodie, Miss S. Mitchinson and Mrs D. Towell for the typing.

Araki
,
M.
&
Okada
,
T. S.
(
1978
).
Effects of culture media on the ‘foreign’ differentiation of lens and pigment cells from neural retina in vitro
.
Devi. Growth & Differ
.
20
,
71
78
.
Clayton
,
R. M.
(
1978
).
Divergence and convergence in lens cell differentiation: regulation of the formation and specific content of lens fibre cells
.
In Stem Cells and Tissue Homeostasis
(ed.
B.
Lord
,
C.
Potten
&
R.
Cole
).
Cambridge University Press
.
Clayton
,
R. M.
(
1979
).
Genetic regulation in the vertebrate lens cell
.
In Mechanisms of Cell Change
(ed.
J.
Ebert
&
T. S.
Okada
).
New York
:
Wiley
.
Clayton
,
R. M.
,
De Pomerai
,
D. I.
&
Pritchard
,
D. J.
(
1977
).
Experimental manipulation of alternative pathways of differentiation in cultures of embryonic chick neural retina
.
Devi. Growth and Differ
.
19
,
319
328
.
Coulombre
,
A. J.
(
1965
).
The eye
.
In Organogenesis
(ed.
R. L. de
Haan
&
H.
Ursprung
), pp.
219
251
.
New York, Chicago, San Francisco, Toronto, London
:
Holt, Rinehart and Winston
.
Crane
,
R. K.
&
Ball
,
E. G.
(
1951a
).
Factors affecting the fixation of C14O2 by animal tissues
.
J. biol. Chem
.
188
,
819
832
.
Crane
,
R. K.
&
Ball
,
E. G.
(
1951b
).
Relationship of C14O2 fixation to carbohydrate metabolism in retina
.
J. biol. Chem
.
189
,
269
276
.
Eagle
,
H.
(
1959
).
Amino acid metabolism in mammalian cell cultures
.
Science, N.Y
.
130
,
432
437
.
Earle
,
W. R.
(
1943
).
Production of malignancy in vitro. IV. The mouse fibroblast and changes seen in the living cells
.
J. Natn. Cancer Inst
.
4
,
165
212
.
Eguchi
,
G.
(
1979
).
‘Transdifferentiation’ in pigmented epithelial cells of vertebrate eyes in vitro
.
In Mechanisms of Cell Change
(ed.
J.
Ebert
&
T. S.
Okada
).
New York
:
Wiley
.
Graymore
,
C. N.
(
1969
).
General aspects of the metabolism of the retina. In The Eye
, vol.
1
,
Vegetative Physiology and Biochemistry
(ed.
H.
Davson
), second edition.
New York and London
:
Academic Press
.
Itoh
,
Y.
(
1976
).
Enhancement of differentiation of lens and pigment cells by ascorbic acid in cultures of neural retinal cells in chick embryos
.
Devi Biol
.
54
,
157
162
.
Itoh
,
Y.
,
Okada
,
T. S.
,
Ide
,
H.
&
Eguchi
,
G.
(
1975
).
The differentiation of pigment cells in cultures and chick embryonic neural retinae
.
Devi. Growth and Differ
.
17
,
39
50
.
Jackson
,
J. F.
,
Clayton
,
R. M.
,
Williamson
,
R.
,
Thomson
.,
I.
,
Truman
,
D. E. S.
&
De Pomerai
,
D. I.
(
1978
).
Sequence complexity and tissue distribution of chick lens crystallin mRNAs
.
Devi. Biol
.
65
,
383
395
.
Lehninger
,
A. L.
(
1972
).
Biochemistry
.
New York
:
Worth
.
Loewenstein
,
W. R.
(
1968
).
Emergence of order in tissues and organs. Communication through cell junctions, implications in growth control and differentiation
.
Devi Biol
. Suppl.
2
,
151
185
.
Lopashov
,
G. V.
(
1963
).
Developmental Mechanisms of Vertebrate Eye Rudiments
. Translated by
J.
Medawar
.
Oxford, etc
.:
Pergamon Press
.
Nomura
,
K.
&
Okada
,
T. S.
(
1979
).
Age-dependent change in the transdifferentiation ability of chick neural retina in cell culture
.
Devi. Growth and Differ
.
21
,
161
168
.
Okada
,
T. S.
(
1976
).
Transdifferentiation of cells of specialised eye tissues in cell culture
.
In Tests of Teratogenicity In Vitro
, pp.
91
105
.
Amsterdam
:
North-Holland
.
Okada
,
T. S.
(
1977
).
A demonstration of lens-forming cells in neural retina in clonal cell culture
.
Devi. Growth and Differ
.
19
,
47
55
.
Okada
,
T. S.
,
Itoh
,
Y.
,
Watanabe
,
K.
&
Eguchi
,
G.
(
1975
).
Differentiation of lens in cultures of neural retinal cells of chick embryos
.
Devi Biol
.
45
,
315
329
.
Okada
,
T. S.
,
Yasuda
,
K.
,
Araki
,
M.
&
Eguchi
,
G.
(
1979
).
Possible demonstration of multipotential nature of embryonic neural retina by clonal cell culture
.
Devi Biol
.
68
,
600
617
.
De Pomerai
,
D. I.
&
Clayton
,
R. M.
(
1978
).
Influence of embryonic stage on the transdifferentiation of chick neural retina cells in culture
.
J. Embryol. exp. Morph
.
47
,
179
193
.
De Pomerai
,
D. I.
,
Pritchard
,
D. J.
&
Clayton
,
R. M.
(
1977
).
Biochemical and immunological studies of lentoid formation in cultures of embryonic chick neural retina and day-old chick lens epithelium
.
Devi Biol
.
60
,
416
427
.
Pritchard
,
D. J.
,
Clayton
,
R. M.
&
De Pomerai
,
D. I.
(
1978
).
‘Transdifferentiation’ of chicken neural retina into lens and pigment epithelium in culture: controlling influences
.
J. Embryol. exp. Morph
.
48
,
1
21
.
Pritchard
,
D. J.
&
Ireland
,
M. J. J.
(
1977
).
A method for relocation of specified regions in tissue culture dishes
.
Experientia
33
,
1120
.
Thomson
,
I.
,
De Pomerai
,
D. I.
,
Jackson
,
J. F.
&
Clayton
,
R. M.
(
1979
).
Lens-specific mRNA in cultures of embryonic chick neural retina and pigmented epithelium
.
Expl Cell Res
.
122
,
73
81
.
Whittaker
,
J. R.
(
1974
).
Aspects of differentiation and determination in pigment cells
.
In Concepts of Development
(ed.
J.
Lash
&
J. R.
Whittaker
), pp.
163
178
.
Stamford, Connecticut
:
Sinauer Associates Inc
.
Yamada
,
T.
(
1976
).
Dedifferentiation associated with cell-type conversion in newt lens regenerating system: a review
.
In Progress in Differentiation Research
(ed.
N.
Muller-Bérat
et al. 
), pp.
355
360
.
Amsterdam
:
North-Holland
.