/. Embryol. exp. Morph. Vol. 62, pp. 47-62, 1981
Printed in Great Britain © Company of Biologists Limited 1981
47
Transdifferentiation of chicken embryo
neural retina into pigment epithelium:
indications of its biochemical basis
ByD. J. PRITCHARD
From the Department of Human Genetics
University of Newcastle upon Tyne
SUMMARY
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.
INTRODUCTION
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,
Author's address: Department of Human Genetics, The University, 19 Claremont Place,
Newcastle upon Tyne, NE2 4AA, U.K.
48
D. J. PRITCHARD
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 pigmentcell precursors, or 'potential pigment cells', is directly proportional to bicarbonate 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 bicarbonate-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 TCAcycle 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.
MATERIALS AND METHODS
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, 100 i.u./ml penicillin and 100/tg/ml streptomycin
(all materials from Gibco Europe). The cells were inoculated at 7 x 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
Transdifferenlialion of chick embryo neural retina
49
sodium hydroxide) at 16-7 (LH), 8-4 (LI) or 1-7 mM (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 mM sodium lactate,
while the others continued receiving standard EMEM. These 'late 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.
RESULTS
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.
Minute and large cells
When released by trypsinization, the undistinguished cells and their derivatives
adopt a spherical shape of diameter 10-20/tm, whereas those which have not
expanded become spheres of 2-5 /im diameter (Fig. 1 A). 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.
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.
50
D. J. P R I T C H A R D
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 /im.
Transdifferentiation of chick embryo neural retina
20
30
Time (days)
Fig. 2. Growth curves of large, minute and total neural retina cells in a control
set of cultures grown in standard EMEM. Each point represents the mean haemocytometer count of cells harvested from three flasks. # , Total; O, large; #, minute.
00
0-4
0-8
1-2
Concentration of supplement (g/1)
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.
51
52
D. J. PRITCHARD
Transdifferentiation of chick embryo neural retina
53
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).
Sequence of differentiative events
Time-lapse photographic analysis of cultures raised in standard EMEM
revealed the sequence of morphological changes which occur as undistinguished 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 jiim, 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/*m. These cells then subdivide a further two or three times to produce
potential pigment cells of about 12/mi 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.
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.
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).
54
D. J. PRITCHARD
Table 1. The effects of the supplements upon pigmentation
Mean area
No. of pigment Pigmentation initia- (mm2) of pigment
colonies (S.E.) tion frequency* (S.E.) colonies f (S.E.)
Control
Succinate
Low
Intermediate
High
Malonate
Low
Intermediate
High
Lactate
Low
Intermediate
High
102 (19-2)
15 (4-1)
P < 002
59 (20-2)
50 (3-7)
P < 005
133 (25-3)
0
P < 002
0
P < 002
71 (5-2)
6(3-8)
P < 002
0
P < 002
12-2(1*15)
5-0(1-36)
10-3 (1-70)
2-9 (0-43)
18-5(2-15)
14-8 (2-20)
2-9 (0-35)
1-7(0-58)
P < 005
270 (2-60)
P < 002
0
P < 002
0
P < 002
2-7(1-57)
14-0(1-74)
11-8(8-75)
t
0
P < 002
0
P < 002
1-2(0-18)
P < 005
~ 1-2
probably significant
0
P < 002
* Pigment colonies per cm2 of combined area of pigment and potential pigment cells.
t Area occupied by pigment cells divided by number of pigment colonies.
t This figure could not be calculated as there were no potential pigment cells.
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 ofpigmentation
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.
Transdifferentiation of chick embryo neural retina
55
Fig. 5. Diagram to show relative percentage areas of vessels occupied by undistinguished cells (D), pavement epithelium (EH) potential pigment ( ^ ) and pigment
cells ( • ) in terminal cultures of neural retina. (A-K) as in Fig. 4.
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.
56
D. J. PRITCHARD
-20
0-4
0-8
1-2
Concentration of supplement (g/1)
00
E
ith elii
-t
ote tia
\
c
•
\
.4
E
pave
S,
1-6
B
80 ~ #
"o D.
o
60
<D
_
40 -
o
ca
20 -
o. <cL>
H
<u
D,
00
l~
0 1
00
—
4
*""^ -^» ^^
i
i
1
0-4
0-8
1-2
Concentration of supplement (g/1)
•
1-6
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.
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.
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, 6 A). Comparison of Figs 6 A 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
Transdifferentiation of chick embryo neural retina
57
Table 2. The effect oflactate on mature cultures
Cultures were exposed to medium containing 16-7 mM sodium lactate at day 56
and assessed twenty days later.
Measure
Control
Lactatetreated
(S.E.)
(S.E.)
Significance
Cell sheet to pavement*
48%
43%
N.S.
(46)
(6-2)
Pavement to pot. pig.*
89%
75%
P < 005
(13)
(53)
Pot. pig. to pigment*
56%
50%
N.S.
(30)
(32)
No. of pigment
128
83
P < 002
colonies
(116)
(84)
Pigmentation initiation
121
10-5
N.S.
(17)
frequency
(0-69)
Mean area of
4-67
4-90
N.S.
pigment colonies (mm2)
(0-38)
(0-68)
* The mean percentage area of the first eel1 type which reached the next stage of differentiation.
0-4
0-8
1-2
Concentration of supplement (g/1)
1-6
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.
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.
58
D. J. PRITCHARD
^Vl
VI
Transdifferentiation of chick embryo neural retina
59
DISCUSSION
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.
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. 6 A). 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 differentiate 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-compensate at low levels of supplementation.
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.
60
D. J. PRITCHARD
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 neighbouring 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 lowlevel 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.
Transdijferentiation of chick embryo neural retina
61
It is with pleasure that I 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. Dunwoodie, Miss S. Mitchinson and Mrs D. Towell for the typing.
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