/ . Embryo/, exp. Morph. Vol. 47, pp. 179-193, 1978
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Printed in Great Britain © Company of Biologists Limited 1978
Influence of embryonic stage
on the transdifferentiation of chick neural
retina cells in culture
By D. I. DE POMERAI 1 AND R. M. CLAYTON
From the Institute of Animal Genetics, University of Edinburgh, Scotland
SUMMARY
Neural retina cells from chick embryos up to 15 days of incubation can transdifferentiate
in culture into both lentoids and pigment cells. Some transdifferentiation into pigment cells
but none into lentoids was found in cultures of 17-day embryonic neural retina. No transdifferentiation occurred in cultures of neural retina from embryos immediately before
hatching. In general, lentoids and pigment cells develop more rapidly and in greater numbers
in cultures of neural retina from the earlier embryonic stages, and lens-specific crystallins
also appear earlier and accumulate in greater amounts in these cultures. Delta crystallin
accumulation is much greater in transdifferentiating cultures of early embryonic neural
retina, whereas a and /? crystallins become proportionately more prominent in cultures of late
embryonic neural retina. Traces of a and/? but not 8 crystallin are detectable in 60-day cultures
of 17-day embryonic neural retina. Analogies between these results and the ontogeny of crystallin polypeptides in lens cells in vivo are discussed.
INTRODUCTION
Conversion of one specialized cell type into another occurs both in vivo and
in vitro in the tissues of the vertebrate eye. The best-studied example of this
in vivo is Wolffian lens regeneration from the dorsal iris epithelium in urodeles
(reviewed by Yamada, 1977), though lens cells can also be derived from several
other eye tissues in vivo (reviewed by Clayton, 1978 a). In early chick embryos
the neural retina can regenerate from the underlying pigmented epithelium
(Coulombre & Coulombre, 1965).
During cell culture of eye tissues in vitro, lens cells may differentiate as
rounded lentoid bodies composed of elongated, fibre-like cells wrapped around
each other. Morphologically and ultrastructurally, lentoid cells resemble true
lens fibres, and they also contain high concentrations of the lens-specific
crystallins. Such lentoids may be derived from newly hatched chick lens epithelial (LE) cells in mass or clonal culture (Okada, Eguchi & Takeichi, 1971;
Okada, Eguchi & Takeichi, 1973), from mass or clonal cultures of 8-day
embryonic chick pigmented epithelium (PE) cells (Eguchi & Okada, 1973),
1
Author's present address: Department of Zoology, University of Nottingham, University
Park, Nottingham NG7 2RD, U.K.
180
D. I. DE POMERAI AND R. M. CLAYTON
from mass cultures of neural retina (NR) cells from 8-day or 3|-day chick
embryos (Okada, Itoh, Watanabe & Eguchi, 1975; Araki & Okada, 1977), and
also from mass cultures of human foetal NR cells (Okada et al. 1977). In the
case of LE cell cultures, lentoid development may be taken to reflect
the normal process of fibre differentiation. In embryonic lens fibres,
8 crystallin predominates greatly over the a and /? crystallins; later,
8 crystallin declines in importance, and it is far less prominent in lens fibres
laid down after hatching (Rabaey, 1962; Genis-Galvez, Maisel & Castro, 1968;
Truman, Brown & Campbell, 1972). This in vivo trend is reflected under cell
culture conditions in vitro, since embryonic LE cultures accumulate more
8 crystallin than do cultures of LE cells from newly hatched chicks (de Pomerai,
Clayton & Pritchard, 1978). However, lens cells do not develop in vivo from
embryonic chick neural retina or pigmented epithelium. The process by which
these abnormal conversions can be effected under in vitro cell culture conditions
has been termed transdifferentiation (Okada et al. 1975; Okada, 1976). Typically, prolonged culture periods are required (4-5 weeks for NR cultures,
6-10 weeks for PE cultures), during which time the cells will divide many times.
Whether the transdifferentiation of NR cells might result from selection of a
small sub-population of undifferentiated stem cells remains unclear, but in the
case of PE cells it has been shown that clones derived from single pigmented
cells can give rise to lentoid bodies (Eguchi & Okadi, 1973). Transdifferentiation
of NR cells into pigmented epithelium may also occur, requiring some 3-4 weeks
of culture (Itoh, Okada, Ide & Eguchi, 1975), and normally this process accompanies lentoid development in mass cultures of embryonic NR cells. One or
both of these transdifferentiation pathways may be promoted by particular
culture conditions in vitro, while other conditions suppress one or both pathways completely (Okada, 1976; Itoh, 1976; Clayton, de Pomerai & Pritchard,
1977; Clayton, \91%a,b). The production of lens-specific crystallins in the
lentoids formed by transdifferentiation is of particular interest, and these
proteins are well-characterized both biochemically and immunologically (reviewed by Clayton, 1974; Thomson et al. 1978#). We have previously shown
that mature lentoid-containing cultures from 8-day embryonic NR do not
contain the high levels of 8 crystallin characteristic of embryonic lens fibres,
but rather show certain similarities to the cortical fibres of adult chickens in
terms of their crystallin composition (de Pomerai, Pritchard and Clayton, 1977).
Araki & Okada (1977) have recently reported that lentoid formation is much
faster and 8 crystallin accumulation much greater in cultures from 3|-day
embryonic chick NR, as compared with their 8-day counterparts. We now
extend this type of analysis by comparing the patterns of crystallin accumulation
during transdifferentiation of NR cells from a variety of embryonic stages. The
results show a stage-related progression both in terms of the capacity to transdifferentiate, and in the crystallin composition of transdifferentiated lens fibres.
Transdifferentiation of chick neural retina
181
MATERIALS AND METHODS
Fertile eggs were obtained from D. B. Marshall (Newbridge, Midlothian) and
the Poultry Research Centre (Roslin, Midlothian). Sources of chemicals and
apparatus are given below where appropriate.
(1) Cell culture
Neural retina was dissected, cleaned and dissociated with trypsin as described
(de Pomerai et al. 1977; Okada et al. 1975). For 6-day embryos the entire dissection was performed under a dissecting binocular microscope; for 15-day and
later embryos, great care was required to separate the neural retina from the
closely adhering pigmented layer beneath it. The size of inoculum varied from
106 cells per dish in the case of 6-day embryonic NR up to about 108 cells per
dish in the case of 17-day embryonic NR. These initial cell densities were chosen
so that the cells which stuck down and survived were able to form large confluent patches within the first 10-15 days of culture. Very few cells stuck down
in the case of prehatching (21-day embryonic) NR, even with inocula as high as
109 cells per dish. In all cases the cells were established and grown in minimal
essential medium containing Earle's salts, supplemented with 10% foetal calf
serum, lOOi.u./ml penicillin, 100/tg/ml streptomycin, 2mM L-glutamine (all
from GIBCO-Biocult Ltd., Paisley, Scotland) and 0-22% NaHCO 3 . Cultures
were maintained at 37 °C in a humid atmosphere of 95% air:5% CO2, the
medium (4 ml per 6x1-5 cm tissue culture dish) being changed every 2-3 days.
(2) Gel electrophoresis
Cultures at various stages were washed several times in physiological saline
and the cell sheet homogenized in a small volume of saline containing 20 HIM:
/i-mercaptoethanol. The homogenate was centrifuged at 10000 g for 20 min to
remove cell debris and the supernatant frozen in liquid N 2 .
Aliquots containing 30-100 /tg protein were analysed on SDS-polyacrylamide
gradient slab gels as described previously (Thomson et al. 19786). Other aliquots
were analysed by haemagglutination inhibition assays (see (4) below).
(3) Antisera
The antisera against purified a and 8 chick crystallin fractions were prepared
as described previously (de Pomerai et al. 1977), and specific antisera against
nine individual /? crystallin fractions were those characterized fully by Clayton &
Truman (1974). For the purposes of the present work, an antiserum with broad
anti-/? specificity was required, and this was obtained by pooling samples of all
nine specific anti-/? sera. Antisera against a and 8 crystallin were further purified
by means of columns containing respectively /? + # and pure 8 crystallins bound
to CNBr-activated sepharose 4B (M. Jones & R. M. Clayton, in preparation).
These antisera were both monospecific as judged by immunoelectrophoresis
(de Pomerai et al. 1977).
182
D. I. DE POMERAI AND R. M. CLAYTON
(4) Haemagglutination inhibition assays
The haemagglutination inhibition assays used here are based on the method of
Evans, Steel & Arthur (1974), as modified by de Pomerai et al. (1977). Originally
we used sheep red blood cells coated with total day-old chick lens protein as
indicator cells (de Pomerai et al. 1977), but we have since found that these
do not always agglutinate well with anti-/? or anti-a sera, owing to the preponderance of 8 crystallin on the surfaces of the indicator cells. In the present
work we have used indicator sheep red blood cells coated with adult chick
cortex crystallins (where a and /? predominate over 8) when assaying for chick a
and /? crystallins. Indicator cells coated with total day-old chick lens protein
were used as previously when assaying for 8 crystallin.
After incubation of twofold serial dilutions of the test sample (8 /tl) with
8 jtd antiserum (at such a dilution that it would just agglutinate the standard
aliquot of indicator cells), the indicator cells (8 /d of a 2 % suspension) were
added and the mixture left to stand for several hours. The endpoint dilution at
which the test sample ceased to inhibit agglutination was then noted and compared with that for identical assays on a known standard containing 0-275 mg/
ml of total day-old chick lens protein. The concentration of a particular crystallin class (a, /? or 8, according to the antiserum used) in the test sample,
relative to its concentration in the total day-old lens, is given by the equation:
endpoint dilution for test sample x 0-275 x 100
endpoint dilution for standard x protein concentration of test sample (mg/ml)
From gel integration and other techniques, we estimate that total day-old chick
lens crystallins comprise approximately 60 % 8 crystallin, 25 % /? crystallin and
15% a crystallin. Using these values as conversion factors, the concentration
of each crystallin class in the test sample may be expressed as a percentage of the
total soluble protein.
The protein concentration in each test sample was estimated by means of
scaled-down Lowry assays (Lowry, Rosebrough, Farr & Randall, 1951), using
total day-old chick lens protein as a standard. All haemagglutination inhibition
assays were performed at least in duplicate, and mean values from several sets
of assays are plotted in Figs. 3-5.
RESULTS
(1) Development in culture of neural retina cells from various
embryonic stages
Figure 1 shows phase-contrast photographs of typical features during the
development of 6-, 8-, 12-, 15-, 17- and 21-day embryonic chick NR cells in
culture. In all but the last of these cultures, extensive confluent sheets of cells
were formed within 10-15 days. Very different inoculum sizes (see legend to
Fig. 1) were required to achieve this, as the earlier embryonic cells stuck down
Transdifferentiation of chick neural retina
183
more readily and grew more vigorously than their later counterparts. In the
case of 21-day embryonic (prehatching) NR, very few cells stuck down even at
the highest densities attempted, and these grew very poorly. Groups of neuroblast-like cells were found overlying the cell sheet after about 8 days in the case
of NR cultures from 6-, 8-, 12-, 15- and 17-day embryos; in some cases these
groups were interconnected by long processes, which seemed especially welldeveloped in the case of 8-day material (see also Okada et al. 1975; Okada,
1976). By about 20 days, most cultures contained groups of small polygonal
cells which have been designated 'prepigment cells' (Pritchard, de Pomerai &
Clayton, 1978); these were especially marked in cultures from 6- and 17-day
material but were absent in cultures from 21-day embryonic NR. By 30 days
of culture both lentoids and patches of melanin-containing pigment cells were
present in the 6-, 8-, 12- and 15-day embryonic NR cultures, but only pigment
cells developed in cultures from 17-day material. Lentoids first appeared at 23,
25, 25 and 28 days respectively in the case of 6-, 8-, 12- and 15-day embryonic
NR cultures. Differences in the frequency of lentoids and pigment patches
became accentuated between 30 and 50 days of culture. Both occurred sparsely
in NR cultures from 15-day embryos, more frequently in NR cultures from
12-day embryos, and abundantly in cultures from 8- and 6-day material. In
45-day cultures from 6-day embryonic NR, the larger lentoids were connected
together by ridges of cellular material, the underlying cell sheet being almost
entirely converted into pigmented cells (Fig. 2). No signs of transdifferentiation
were seen in the cultures from 21-day embryonic NR, while in cultures from
17-day material, only transdifferentiation into pigment cells was observed.
A very few structures which might represent small lentoids were observed at
around 50 days in these latter cultures (Fig. 1; 17-day NR, 40-50 days, B),
but only traces of crystallin were detectable even 10 days later than this (see
below).
(2) Crystallin composition during lentoid development in cultures of
neural retina cells from various embryonic stages
The foregoing observations on the time of appearance and frequency of
lentoids are reflected by the patterns of crystallin accumulation in these NR
cultures (Fig. 3). The rate of crystallin accumulation in culture falls off with
increasing developmental age of the NR starting material; thus some 60% of
the total soluble protein is crystallin in the case of 52-day cultures from 6-day
embryonic NR, as compared to 10% in the case of 15-day material after the
same period in culture (Fig. 3). The proportions of the main crystallin classes
(a, /? and 8) change as the total crystallin content rises; this is shown in Fig. 4
for each time-point used in Fig. 3. Generally speaking, 8 crystallin tends to
increase in prominence during the culture period while a crystallin declines,
with /? crystallin remaining approximately constant in the case of late embryonic
NR cultures, or decreasing along with a in the case of early embryonic NR
184
D. I. DE POMERAI AND R. M. CLAYTON
(a)
Days in culture
8-10 day
18-21 day
28-31 day (A)
NR from
6-day emb.
8-day emb.
12-day emb.
15-day emb.
17-day emb.
21-day emb.
FIGURE 1
Development in culture of neural retina cells from different embryonic stages. NR
cultures were established as described in Methods, using inocula of 106, 5 x 106,107,
5x 107, 10s and 109 cells per dish for 6-, 8-, 12-, 15-, 17- and 21-day embryonic
material, respectively. These cultures were examined by phase-contrast microscopy
at 3-day intervals, and photographs of typical features are shown in Fig. 1 for each
embryonic age at 8-10 days, 18-21 days, 28-31 days and 45-50 days of culture.
Two fields are shown for each of the last two time points, in order to illustrate the divergent modes of development towards pigment cells (column A) and lentoids
(column B) respectively. Where no lentoids developed (17- and 21-day embryonic
NR cultures), photographs of putative neuronal cells and processes have been substituted in column B.
Transdifferentiation of chick neural retina
185
(b)
Days in culture
28-31 day(B)
45-50 day (A)
45-50 day (B)
NRfrom
6-day emb.
8-day emb
12-day emb
FIGURE
\b
cultures. The proportion of total crystallin represented by 8 is highest (up to
80 %) in cultures from 6-day embryonic NR, progressively lower in cultures
from 8-day material (up to 60%) and 12-day material (up to 45 %), and lowest
(only 30 %) in cultures from 15-day embryonic NR. The proportion of /? crystallin is small (5-15%) in cultures from 6-day material, varies from 20 to 40%
in cultures from 8-day material, but remains relatively constant (25-30%) in
186
D. T. DE POMERAI AND R. M. CLAYTON
Fig. 2. Lentoids and pigment cells in a 45-day culture of 6-day neural retina.
A composite photograph of several adjacent fields showing lentoids and pigment
cells in a 45-day culture from 6-day embryonic NR. Phase contrast microscopy and
cell culture as for Fig. 1.
cultures from later embryonic NR. The proportion of a crystallin is greatest
(about 40 %) in cultures from 15-day embryonic NR, intermediate in the case of
12-day material (25-35 %) and 8-day material (20-30%), and lowest (10-15 %)
in cultures from 6-day NR. The levels of a, /? and 8 crystallin are separately
compared in Fig. 5 for cultures 6-, 8-, 12-, 15- and 17-day embryonic NR. The
rates of accumulation of all three crystallin classes are greater in cultures of
early than of late embryonic NR, but this trend is much more pronounced in
the case of 8 crystallin. Thus at 52 days 8 crystallin reaches 20-fold higher levels
in cultures from 6- as compared to 15-day material, whereas for a or /? crystallins this difference is only about twofold. The onset of detectable crystallin
accumulation precedes the appearance of lentoids in the case of NR cultures from
6- and 8-day embryos (cf. de Pomerai et al. 1977), but in cultures from 12- and
15-day material, crystallins only reach detectable levels when the lentoids start
to appear (Fig. 5). In cultures of 17-day embryonic NR, no crystallins were
detectable after 38 days in culture, but traces (0-2-0-6 % of the total soluble
protein) of y^ and a but not 8 crystallins were found by 60 days (Fig. 5).
Figure 6 shows the pattern of bands obtained by SDS-polyacrylamide
gradient gel electrophoresis of soluble proteins extracted from NR cultures.
In contrast to the gel system previously employed (de Pomerai et al. 1911), this
method gives good resolution in the 8 crystallin region (M.W. 45000; Piatigorsky,
Zelenka & Simpson, 1974). Although a minor component comigrating with
Transdifferentiation of chick neural retina
60 .5
1
50 -
4
I!
<>-
b
30-
c°
2010 0
26 45 52
Days
100
3C
3B
3 A
26 35 42 56
Days
4B
4 A
187
3D
35 42 50
Days
4C
32 45 52
Days
4D
9080 70 60 50 40 30 20 10 0
26 45 52
Days
26 35 42 56
Days
32 42 50
Days
32 45 52
Days
Fig. 3. Quantitation of total crystallins in cultures of neural retina from different
embryonic stages. At the indicated time points, cultures from 6-, 8-, 12- and 15-day
embryos were washed and the soluble proteins extracted as described in Methods.
Haemagglutination inhibition assays were used to quantitate the amounts of a,
total ft and 8 crystallins in each extract. These amounts were expressed as percentages
of total soluble protein (see Methods), and the total (a +/?T0T + #)% is shown as a
bar for each time point in Fig. 3. Part A, levels of crystallin in cultures of 6-day
embryonic NR; Part B, levels of crystallin in cultures of 8-day embryonic NR;
Part C, levels of crystallin in cultures of 12-day embryonic NR; Part D, levels of
crystallin in cultures of 15-day embryonic NR.
Fig. 4. Proportions of a, ft and 8 crystallins in cultures of neural retina from different
embryonic stages. The respective amounts of a, ft and 8 crystallins (see legend to
Fig. 3) are here expressed as percentages of the total crystallin present at each stage
in each culture series. For each bar, the total crystallin is taken as 100%, and the
proportions of a, ft and 8 crystallins are shown by the extent of cross-hatching,
of stippling, and of horizontal line shading, respectively. Each bar in Fig. 4 is placed
immediately under the corresponding bar for Fig. 3. Part A, proportions of a, ft and
8 crystallins in cultures of 6-day embryonic NR; Part B, proportions of a, ft and 8
crystallins in cultures of 8-day. Part C, proportions of a, ft and 8 crystallins in cultures of 12-day embryonic NR; Part D, proportions of a, ft and 8 crystallins in
cultures of 15-day embryonic NR.
188
D. I. DE POMERAI AND R. M. CLAYTON
50 -
A
-
40 -
/
soliuble pi"otein
30 -
-2'
/
20 -
/ /
f
10
1
8 -
//
! ,
6 -
I/
///
4 2 0
^ zv. .
0 JO 20 30 40 50 60
l
1
1
1
1
1
0 -
1
0
10 20 30 40 50 60
Days in culture
0 10 20 30 40 50 60
Fig. 5. Quantitation of a, ^ and #crystallins in cultures of neural retina from different
embryonic stages. The levels of a, ft and tfcrystallins in NR cultures were determined
by haemagglutination inhibition assays, and expressed as percentages of the total
soluble protein at each time point. In addition to those points corresponding to the
data in Figs. 3 and 4, additional points are given for the early stages of culture in
each series, and also for 17-day embryonic NR cultures. • — • , 6-day embryonic
NRcultures; # — # , 8-day embryonic NR cultures; A""A, 12-day embryonic NR
cultures; • - • - • , 15-day embryonic NR cultures; T—T, 17-day embryonic NR
cultures. Part A, quantitation of 8 crystallin; Part B, quantitation of total (i
crystallins; Part C, quantitation of total acrystallins.
8 crystallin is present even in very young cultures (e.g. 6-day cultures from 8-day
embryonic NR), the later increase in prominance of this band (e.g. in 35- and
56-day cultures from 8-day embryonic NR) is probably due to the rise in
genuine 8 crystallin (reaching 20% of the total soluble protein in 56-day
cultures from 8-day NR; see Fig. 5). Beta crystallins are poorly resolved from
neural retina components of similar molecular weight, but two bands comigrating with the a crystallins can be seen clearly in the later cultures from all
embryonic stages. Figure 7 compares the protein compositions of 42-day
cultures from 6-, 8- and 12-day embryonic NR. The relative amounts of a and
8 crystallins as shown by immunological methods (Fig. 5) are here clearly
paralleled by the relative intensities of the putative cc and 8 crystallin bands.
189
Transdifferentiation of chick neural retina
6-day emb. NR
r—
S
10
26
52
8-day emb. NR
6
12 26 35 56
f
Delta
Betas
1 wm • • |T* J C
Alphas C = #
12-day emb. NR
6 14 26 35 42 50
15-day emb. NR
Fig. 6. Protein composition of cultures of neural retina from different embryonic
stages. Soluble proteins were extracted from NR cultures at various stages and
samples (30-100 fig) were run on SDS polyacrylamide gradient slab gels as described
in Methods. On each slab gel, two samples of total day-old lens protein were also run
as a standard, and the crystallin bands were identified according to Thomson et al.
(1978 a, b). Since the gels photographed in Fig. 6 came from a number of different
runs, the positions of the main a and 8 bands are marked by arrows for each slab,
and only in one case is the day-old protein standard (S) also shown. Typical protein
profiles are shown for 6-, 8-, 12- and 15-day embryonic NR cultures over a range of
time points from 6 to 56 days of culture; for each sample, the number of days in
culture is given in arabic numerals above the corresponding gel slot. Slots from the
same gel are grouped together.
EMB 47
190
D. I. DE POMERAI AND R. M. CLAYTON
12
8
6
S
-Delta
Betas
Alphas
Fig. 7. Protein composition of 42-day cultures of 6-, 8- and 12-day embryonic NR.
80 fig samples of soluble protein from 42-day cultures of 6-, 8- and 12-day embryonic
NRwere run on an SDS polyacrylamide gradient slab gel and stained as described in
Methods. Total day-old chick lens proteins (40/<g) were also run on the same slab,
and the resulting crystallin profile (S) is shown alongside. Arrows indicate the
positions of a and 8 crystallin bands, and the j3 crystallin region is also shown.
Arabic numerals here indicate the number of days of embryonic development for the
starting NR, all cultures being incubated for the same period (42 days) in vitro.
DISCUSSION
The transdifferentiation of embryonic chick NR cells in culture into pigmented
epithelium and lens fibres (lentoids) shows several progressive changes according
to the developmental age of the starting material. The range of developmental
ages tested in the present work (6- to 21-day embryonic NR) may be extended
back to earliei stages by comparing our results with the data of Araki & Okada
(1977) on transdifferentiation in cultures from 3|-day embryonic chick NR.
Several differences between cultures from 3|-day NR and from 8-day NR were
pointed out by Araki & Okada (1977), and our present data indicate that these
represent consistent trends. In particular, early embryonic neural retina cells
grow more vigorously and require less time for transdifferentiation into both
lens and pigment cells, than do their later embryonic counterparts (see Results).
Moreover, in cultures from early embryonic NR, almost all the cells transdifferentiate either into lentoids or pigmented epithelium (for 3|-day material,
see Araki & Okada, 1977; for 6-day material, see Fig. 2), whereas transdifferentiation events of either type are much rarer in cultures from later (e.g.
15-day) embryonic NR. The case of 17-day embryonic NR is particularly
interesting; here, transdifferentiation into lens cells is almost completely suppressed (no convincing lentoids and very low levels of crystallin; Figs. 1 and 5),
Transdifferentiation of chick neural retina
191
but transdifferentiation into pigment cells is still possible and may even be
slightly enhanced in comparison with cultures of 15-day NR (Fig. 1). Neither
type of transdifferentiation was observed in cultures from prehatching (21-day
embryonic) NR cells (Fig. 1), nor in cultures from 1-, 2- and 7-week adult
chicken NR cells (de Pomerai, unpublished observations).
Several consistent trends are also observable in the crystallin composition
of lens cells formed by transdifferentiation in cultures of NR from different
developmental stages.
The accumulation of crystallin is much faster and starts much sooner in the
case of NR cultures from early embryos (for 3|-day material, see Araki &
Okada (1977); for 6-day and later stages, see Results and Fig. 3). The content of
8 crystallin is also much higher in cultures of early embryonic NR. The data of
Araki & Okada (1977) indicate that 8 predominates over a crystallin by approximately 20 to 1 in 30-day cultures from 3|-day material. In 45- and 52-day
cultures from 6-day embryonic NR the ratio of 8:CL crystallin is about 7:1
(Fig. 4), and this 8:a ratio further declines to about 3:1, 2:1 and 1:1
respectively in 50- to 56-day cultures from 8-, 12- and 15-day embryonic NR
(Fig. 4). Araki & Okada (1977) gave no data on the /? crystallin content of cultures from 3|-day embryonic NR, but one might expect the value to be rather
low from the trend seen in Fig. 4, where /? comprises only 5-8 % of the total
crystallin in cultures from 6-day embryonic NR after 45-52 days in culture.
This predominance of 8 crystallin in cultures of early embryonic NR, and
the increasing contribution of a and {} crystallins in cultures from later embryonic
NR (Fig. 4), are paralleled by a similar trend in cultures of lens epithelial (LE)
cells from various stages in chick development. Here, 8 crystallin accumulation
was greatest in cultures of 12-day embryonic LE cells, intermediate in cultures of
15-day embryonic LE cells, and poorest in cultures of newly hatched LE cells
(de Pomerai et al. 1978). A correlation between the growth rate and crystallin
composition of these LE cultures was indicated, and experiments were suggested
which could test for any causal link between these two parameters (Clayton,
1978b; de Pomerai et al. 1978). Differential growth rates might also influence
the crystallin composition of lens cells formed in transdifferentiating NR
cultures. It should be noted that LE and NR cultures set up from the same
stage produce quite different proportions of the main crystallin classes (e.g.
high 8 in 12- or 15-day embryonic LE cultures, but low 8 in 12- or 15-day
embryonic NR cultures). Although the amounts of 8 crystallin produced in
LE cultures from different stages may reflect the normal differentiation into
fibres which those LE cells would undergo in vivo (de Pomerai et al. 1978), this
is clearly not the case for NR cultures. A rigorous comparison of growth curves
for cultures of both LE and NR cells from different developmental stages would
help to establish the correlation between growth rate and crystallin composition,
but in itself such a study could neither prove nor disprove the hypothetical link
between them. One possible source of evidence on this score would be an
13-2
192
D. I. DE POMERAI AND R. M. CLAYTON
investigation of the effect on crystallin composition of a variety of culture
conditions (Okada, 1976; Itoh, 1976; Clayton et al. 1977) which affect the
growth rate of both types of culture. Preferably, both growth-promoting and
growth-inhibiting conditions should be tested to see whether these lead to an
increase and a decrease respectively in the proportion of £ crystallin synthesized.
The authors would like to thank D. B. Marshall Ltd. and the Poultry Research Centre for
supplying fertile eggs; Drs I. Thomson, D. J. Pritchard, P. G. Odeigah, J. F. Jackson, D. E. S.
Truman and J. C. Campbell for useful discussions and suggestions during the course of this
work; and the M.R.C. and C.R.C. for grants in support of this research. D. de P. is in
receipt of an M.R.C. postdoctoral training fellowship.
REFERENCES
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neural retinal cells of early chick embryos. Devi Biol. 60, 278-287.
CLAYTON, R. M. (1974). Comparative aspects of lens proteins. In The Eye (ed. H. Davson &
L. T. Graham, Jnr.), vol. 5, pp. 115-180. New York and London: Academic Press.
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 Homeostasis (ed. B. I. Lord, C. S. Potten & R. J. Cole). Cambridge University Press. (In the
Press.)
CLAYTON, R. M. (19786). Genetic regulation in the vertebrate lens cell. In Mechanisms of
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