/. Embryol. exp. Morph. Vol. 48, pp. 1-21, 1978
Printed in Great Britain © Company of Biologists Limited 1978
'Transdifferentiation' of chicken neural
retina into lens and pigment epithelium in
culture: controlling influences
By D. J. PRITCHARD 1 , R. M. CLAYTON 2
AND D. I. DE POMERAP
From the Institute of Animal Genetics, University of Edinburgh
SUMMARY
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.
INTRODUCTION
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,
1
Author's address: Department of Human Genetics, 19 Claremont Place, Newcastle upon
Tyne, NE2 4AA, U.K.
2
Authors' address: Institute of Animal Genetics, University of Edinburgh, King's
Buildings, West Mains Road, Edinburgh EH9 3JN, Scotland, U.K.
2
D. J. PRITCHARD, R. M. CLAYTON AND D. I. DE POMERAI
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.
MATERIALS AND METHODS
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 etal 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,
lOOi.u./ml penicillin and 100/Ag/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
Transdifferentiation
of neural retina
3
& 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 % CO 2 :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 EM.EM 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.
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 x 106 cells in 5 ml of medium and
bottles at around 7 x 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 bcttles 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
D. J. PRITCHARD, R. M. CLAYTON AND D. I. DE POMERAI
Table 1. Concentrations of sodium, potassium and bicarbonate ions
in the media as formulated
Concentrations in gram ions per litre
a,
b,
c,
d,
Medium
Gas phase
K
Na
HCO3
EMEM"
EMEMK*
HMEMC
mod. HMEM"
5 % CO2
5% CO,
0%CO 2
5%CO 2
5-3
20-7
5-8
5-8
144-4
118-3
142-7
155-2
26-2
26-2
4-2
16-7
Minimal Essential Medium based on Earle's salts.
As a, but with NaHCO 3 replaced by an equimolar concentration of KHCO 3 .
Minimal Essential Medium based on Hanks' salts.
As c, but with additional NaHCO3 .
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.
RESULTS
(A) Selection of medium
Cultures grown in EMEM consistently developed more pigment than those
in mod. HMEM (Figs. 1 A, IB, 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.
(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. ID). 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.
Transdijferentiation of neural retina
Table 2. Pigmentation developed in mature cultures of neural retina
grown in different media
Medium
Mod.
HMEM
% area of dish occupied by pigmented cells"
Number of pigmented colonies per dish
Mean area of pigmented colonies6
4-8
110
0-85 mm2
EMEM
48
540
1 -66 mm2
a, Estimates are based on a stereological assessment of a typical culture of each class
(see Fig. 1 A, B).
b, Values calculated from the two previous sets of figures.
Abbreviations as in Table 1.
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 oflentoid 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. I E ; cf. Okada et ah 1975). At the same time the multilayers contract
laterally to form thicker masses and lentoids also arise from these (Fig. IF).
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).
6
D . J. P R I T C H A R D , R . M. C L A Y T O N
AND
D . I. D E
POMERAI
Transdifferentiation
r
of neural retina
50
10
20
30
Days of culture
Fig. 2. A typical g r o w t h curve of neural retina cells in E M E M (see Table 1). E a c h
point represents the m e a n h a e m o c y t o m e t e r count of cells harvested from t w o dishes.
(A) Foci of potential pigment cells present; crystallins detectable. (B) Multilayers
present. (C) Melanin accumulates in potential pigment cells. (D) T r u e lentoids
present.
FIGURE 1
(A, B) Unstained terminal cultures of neural retina p h o t o g r a p h e d by transmitted
light. (A) Culture established a n d g r o w n in m o d . H M E M a n d (B) in E M E M (see
Table 1).
( C - J ) N e u r a l retina cultures g r o w n in E M E M p h o t o g r a p h e d u n d e r phase-contrast
illumination. T h e bar represents 100 /im.
(C) A developing colony of pigment epithelium. N o t e pigmented cells at t o p left
a n d bare dish at lower right.
(D) T h e same field as (C), 5 days later.
(E) A m a t u r e colony of pigment epithelium with adjacent lentoids.
( F ) A lentoid in the u p p e r sheet of the multilayer.
( G ) A large aggregate which a p p e a r e d in a late culture.
(H) Small, linked aggregates in an early culture.
(J) Strand of extracellular material in a n old culture.
7
8
D. J. PRITCHARD, R. M. CLAYTON AND D. I. DE POMERAI
Table 3. Pigmentation developed in chilled and control cultures of neural retina
and in cultures grown in medium of high potassium content
% area of dish occupied by
pigmented cells"
Medium
Control6
Chilledc
EMEM
32
34
18
19
7
13
EMEMK
24
17
24
17
14
16
a, Stereological assessments of individual dishes at 60 days.
b, Incubation temperature maintained at 37 °C.
c, Incubation temperature reduced to 28 °C between days 42 and 50.
Abbreviations as in Table 1.
(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 or EMEMK 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.
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
Transdifferentiation of neural retina
9
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
Inoculum
Terminal
density
density
per cm2 (±4%) per cm2 (±4%)
EMEM dish
EMEM bottle
HMEM dish
HMEM bottle
mod. HMEM dish
mod. HMEM bottle
2-4 xlO5
30 xlO 5
2-4 x 105
30 xlO5
2-4 xlO5
30 xlO5
1-7 xlO5
1-2 xlO5
2-2x10*
2-6x10*
3-6 xlO5
3-4 xlO5
Lentoid-like
bodies per cm2
(+S.E.)
77 (4-0)
61 (23-4)
103 (91)
184(22-2)
196(10-8)
400 (35-4)
Approx.
crystallin
content
(%)
15
15
5
5
15
15
Abbreviations as in Table 1.
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).
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).
(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.
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
10
D. J. PRITCHARD, R. M. CLAYTON AND D. I. DE POMERAI
lnccilum
• —
50
40 - HMEM
Mod. HMEM
F.MEM
10
20
o
x
30
3 20
00
00
0-5
1-5
NaHCO., (g/l.)
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.
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.
Transdijferentiation of neural retina
11
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.
(b) Multilayering
Figure 5B reveals the positive relationship between multilayering and cell
density. Multilayering occurs when floor coverage exceeds about 40%.
(c) Lento id 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 /? 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 # crystallin from retinal
12
D. J. PRITCHARD, R. M. CLAYTON AND D. I. DE POMERAI
25
00
10
1-5
NaHCO., (g I.)
00
0-5
10
1-5
N a H C O 3 (g. 1.)
Figures 4 A and B. For legend see opposite page
13
Transdifferentiation of neural retina
0-5
10
1-5
20
NaHCOj (g 1)
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.
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 a and /?
crystallin bands representing a total of about 5 % of the total protein (see Table 4).
The relative concentrations of a crystallin and the 23 000, 24 000-25 000 and
28000 dalton subunits of ft crystallin in the assayable samples and, for comparison, in whole lenses and lens epithelium of day-old chickens, are shown in
Fig. 6.
DISCUSSION
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
2
EMB 48
14
D. J. PRITCHARD, R. M. CLAYTON AND D. I. DE POMERAI
2-5
40
60
Area colonized
£ 20 -
40
60
Area colonized
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 indicates a minimum value
for pigment expression. (B) Extent of the multilayer plotted against percentage area
of the vessel colonized (b = 0-534 ±0-110).
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 ah 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
15
Transdifferentiation of neural retina
EMEM
dish
EMEM
bottle
V7A
Mod HMEM
dish
Mod. HMEM
bottle
J2Z3
Whole lens
(tl)
Lens epithelium
Ui)
m
(<•)
UD
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 - polyacrylamide gels, {a) a crystallin; (b) 23000 dalton subunits of /? crystallin; (c)
24000-25000 dalton subunits of ) crystallin; (d) 28000 dalton subunits of /?
crystallin.
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 lentoidlike 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.
16
D. J. PRITCHARD, R. M. CLAYTON AND D. I. DE POMERAI
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 a or /? 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 explants
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
Transdifferentiation of neural retina
17
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 amonolayer 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.
THE MECHANISM OF TRANSDIFFERENTIATION
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, 1978a, 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
18
D. J. PRITCHARD, R. M. CLAYTON AND D. I. DE POMERAI
(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. 1975) 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 a, /? and 8 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.
Transdifferentiation of neural retina
19
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.
REFERENCES
L. E. (1937). An experimental study of the role of optic-cup and overlying
ectoderm in lens formation in the chick embryo. /. exp. Zool. 75, 41-73.
BARTELMEZ, G. W. & BLOUNT, M. P. (1954). The formation of neural crest from the primary
optic vesicles in man. Contr. Embryol. Cam. Inst. Washington 35, 55-7.1.
BARTH, L. G. & BARTH, L. J. (\914a). Ionic regulation of embryonic induction and cell
differentiation in Rana pipiens. Devi Biol. 39, 1-22.
BARTH, L. J. & BARTH, L. G. (19746). Effect of potassium ion on induction ofnotochordfrom
gastrula ectoderm of Rana pipiens. Biol. Bull. mar. biol. Lab., Woods Hole 146, 313-325.
CAHN, R. D. & CAHN, M. B. (1966). Heritability of cellular differentiation: clonal growth
and expression of differentiation in retinal pigment cells in vitro. Proc. natn. Acad. Sci.,
U.S.A. 55, 106-114.
CLAYTON, R. M. (1975). Failure of regulation of the lens epithelium in strains of fast-growing
chicks. Genet. Res. Camb. 25, 79-82.
CLAYTON, R. M. (1978O). Divergence and convergence in lens cell differentiation: regulation
of the formation and specific content of lens fibre cells. In Stem Cells and Tissue Borneostasis (ed. B. Lord, C. Potten & R. Cole). Cambridge University Press (in the Press).
CLAYTON, R. M. (19786). Genetic regulation in the vertebrate lens cell. In Mechanisms of
Cell Change (ed. J. Ebert & T. S. Okada). Wiley (in the Press).
CLAYTON, R. M., EGucm, G., TRUMAN, D. E. S., PERRY, M. M., JACOB, J. J. & FLINT, O. P.
(1976). Abnormalities in the differentiation and cellular properties of hyperplastic lens
epithelium from strains of chickens selected for high growth rate. /. Embryol. exp. Morph.
35, 1-23.
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.
Develop. Growth & 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 &
Winston.
COULOMBRE, J. L. & COULOMBRE, A. J. (1965). Regeneration of the neural retina in the chick
embryo. Devi Biol. 12, 79-92.
DORRIS, F. (1938). Differentiation of the chick eye in vitro. J. exp. Zool. 78, 385-415.
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. /. natn. Cancer Inst. 4, 165-212.
EGUCHI, G. (1976). 'Transdifferentiation' of vertebrate cells in cell culture. In Embryogenesis
in Mammals, pp. 241-258. C.I.B.A. Symp. 40, Amsterdam, New York: Elsevier.
EGUCHT, G., CLAYTON, R. M. & PERRY, M. M. (1975). Comparison of the growth and differentiation of epithelial cells from normal and hyperplastic lenses of the chick: studies of
in vitro cell cultures. Develop. Growth & Differ. 17, 395-413.
EGUCHT, G. & OKADA, T. S. (1973). Differentiation of lens tissue from the progeny of chick
retinal pigment cells cultured in vitro: a demonstration of a switch of cell type in clonal
cell culture. Proc. natn. Acad., Sci., U.S.A. 70, 1495-1499.
ALEXANDER,
20
D. J. P R I T C H A R D , R. M. CLAYTON AND D. I. DE POMERAI
H. (1965). The stereological approach to quantitative microanatomy. Part 1. Med. bioi
Must. 15, 253-258.
Fox, H. M. & VEVERS, G. (1960). The Nature of Animal Colours. London: Sidgwick &
Jackson Limited.
FUJITA, S. & HORII, M. (1963). Analysis of cytogenesis in chick retina by tritiated thymidine
autoradiography. Arc/is histol. jap. 23, 359-366.
GURDON, J. B. & WOODLAND, H. R. (1970). On the long-term control of nuclear activity
during cell differentiation. In Current Topics in Developmental Biology, vol. 5 (ed. A. A.
Moscona & A. Monroy. New York and London: Academic Press.
HADORN, E. (1966). Dynamics of determination. In Major Problems in Developmental Biology
25th Symp. Soc. Devi Biol. (ed. M. Locke), pp. 85-104. New York: Academic Press.
HANKS, J. H. & WALLACE, R. E. (1949). Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc. Soc. expl Biol. Med. 71, 196-200.
HOLLEY, R. W. (1975). Control of growth of mammalian cells in cell culture. Nature, Lond.
258, 487-490.
ELIAS,
HOLTZER, H., RUBINSTEIN, N. : FELLINI, S., YEOH, G., CHI, J., BIRNBAUM, J. & OKAYAMA, M.
(1975). Lineages, quantal cell cycles, and the generation of cell diversity. Q. Rev. Biophys.
8, 523-557.
ITOH, Y., OKADA, T. S., IDE, H. & EGUCHI, G. (1975). The differentiation of pigment cells
in cultures of chick embryonic neural retinae. Develop. Growth & 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. (in the Press).
A. B. (1955). Mammalian tyrosinase. In Methods in Enzymology, II (ed. S. P.
Colowick & N. O. Kaplan), pp. 827-831. New York: Academic Press.
LOPASHOV, G. V. (1963). Developmental Mechanisms of Vertebrate Eye Rudiments. Translated
by J. Medawar. Oxford, etc.: Pergamon Press.
MACGILLIVRAY, A. J., CAMERON, A., KRAUZE, R. J., RICKWOOD, D. & PAUL, J. (1972). The
non-histone proteins of chromatin: their isolation and composition in a number of tissues.
Biochim. biophys. Acta 111, 384-402.
MCLEAN, B. G. & FINNEGAN, C. V. (1974). Observations on chick embryo lens histogenesis
in vivo and in vitro. Can. J. Zool. 52, 345-352.
OKADA, T. S. (1976). Transdifferentiation of cells of specialized 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. Develop. Growth & 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.
PAUL, J. (1973). Cell and Tissue Culture, 4th edn. Edinburgh and London: Churchill Livingstone.
DE POMERAI, D. I., PRITCHARD, D. J. & CLAYTON, R. M. (1977). Biochemical and immunoJogical studies of lentoid formation in cultures of embryonic chick neural retina and dayold chick lens epithelium. Devi Biol. 60, 416-427.
PRITCHARD, D. J. & IRELAND, M. J. J. (1977). A method for relocation of specified regions
in tissue culture dishes. Experientia 33, 1120.
PRITCHARD, D. J. & CLAYTON, R. M. (1978). Cellular abnormalities of fast-growing chickens:
similarities between retina and lens. Expl Eye Res. 26, 667-670.
LERNER,
REDFERN, N., ISRAEL, P., BERGSMA, D., ROBISON, W. G. JR., WHIKEHART, D. & CHADER, G.
(1976). Neural retinal and pigment epithelial cells in culture: Patterns of differentiation
and effects of prostaglandins and cyclic-AMP on pigmentation. Expl Eye Res. 22, 559-568.
THOMSON, I., WILKINSON, C. E., JACKSON, J. F., DE POMERAI, D. I., CLAYTON, R. M., TRUMAN,
D. E. S. & WILLIAMSON, R. (1978). Characterization of chick lens crystallin mRNA and
its cell free translation during normal development and transdifferentiation of neural retina.
Devi Biol. (in the Press).
WESTON, J. A. (1970). The migration and differentiation of neural crest cells. Adv. Morphogen.
8,41-114.
Transdifferentiation of neural retina
21
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. Stanford, Conn.:
Sinauer Assocs. Inc.
WOLFE, H. G. & COLEMAN, D. L. C1966). Pigmentation. In Biology of the Laboratory Mouse,
2nd edn. (ed. E. L. Green), pp. 405-425. New York, etc.: McGraw-Hill.
YAMADA, T. (1976). Dedifferentiation associated with cell-type conversion in the newt lens
regenerating system: a review. In Progress in Differentiation Research (ed. N. Miiller-Berat
et al., pp. 355-360. Amsterdam: North-Holland.
(Received 28 October 1977, revised 26 June 1978)