/ . Embryol. exp. Morph. Vol. 18, 1, pp. 53-66, August 1967
With 1 plate
Printed in Great Britain
53
A cell proliferation study of the neural retina
in the two-day rat
By S. DENHAM 1
From the Strangeways Research Laboratory, Cambridge
The development of high resolution autoradiography and the availability
of labelled DNA precursors has made possible the detailed analysis of cell
proliferation. Such studies are valuable in the investigation both of normal
growth and also of the effects of radiation and carcinogens.
Wimber (1963) lists three main types of proliferating cell populations: (1)
enlarging population; (2) steady state renewal systems; (3) diminishing populations. Most analytical studies refer to the second group, for example, gut
epithelium and skin, and those of the first category, which includes embryonic
tissues, have received little attention. In the neural retina of the rat cell proliferation continues for about 2 weeks after birth and, for this reason, this was
thought to be a suitable system in which to study cell proliferation during
development. The retina of the 2-day-old rat has the complex cellular constitution of a tissue nearing the end of its proliferative phase. Cell differentiation and
division occur simultaneously and although the population enlarges it does so
at an ever decreasing rate as differentiating cells accumulate (cf. fig. 1, p. 433,
Gliicksmann, 1965). In the experiments to be reported, the cell population of the
retina at 2 days after birth was studied by an autoradiographic method after
administration of tritiated thymidine. The following points were investigated:
(1) the duration of the cell generation cycle and of its constituent phases, i.e.
S: the DNA-synthetic phase, G2: the gap between the end of synthesis and the
onset of prophase, M: the period of visible mitosis and G x : the phase extending
from the end of telophase to the beginning of DNA synthesis (Howard & Pelc,
1953); (2) the timing of differentiation with respect to the phases of the cycle
(3) the size and growth pattern of the proliferative population.
The histogenesis of parts of the central nervous system other than retina has
been studied by several workers after treatment with tritiated thymidine
(Sauer & Chittenden, 1959; Uzman, 1960; Sidman, 1961; Berry, Rogers &
Eayrs, 1964). Kauffman (1966) recently determined generation times for cells
of the embryonic mouse neural tube, and Fujita (1962) made a kinetic study of
1
Author's address: Department of Radiobiology, Institute for Cancer Research, Royal
Marsden Hospital, Sutton, Surrey, U.K.
54
S. DENHAM
the cell population in developing chick retina; both these authors were concerned with a much earlier stage of development than that investigated in the
present experiments. Hicks et al. (1961) made general observations on the
development of embryonic and neonatal rat retina when determining the effects
of ionizing radiation on mammalian embryonic neural tissue, but they did not
analyse in detail the cell generation cycle or growth pattern of the tissue.
MATERIALS AND METHODS
The rats were from a hooded stock bred in the laboratory. Ninety 2-day-old
rats weighing from 5-8 to 6-8 g., fifty 5-day-old animals weighing from 9-5 to
11 -5 g and a few 3- and 4-day rats were used; the 3- and 4-day rats were weighed
at 2 days to ensure that they fell within the normal weight range for this age and
weighed again just before an experiment. Most experiments were started at
10.00 a.m. to avoid effects of diurnal rhythms on the timing of the generation
cycle in the developing retina.
Tritiated thymidine, specific activity 2-8 Ci/mM in the initial experiments and
15 Ci/mM in subsequent experiments, was obtained from the Radiochemical
Centre, Amersham. It was injected subcutaneously beneath the dorsal skin with
an Agla micrometer syringe. In the preliminary experiments one /tCi/gram of
body weight was injected but this was subsequently increased to 3 /*Ci/g to
reduce the exposure time of the autoradiographs to not more than 40 days.
After the necks of the rats had been broken the eyes were removed and fixed
overnight in cold 3:1 ethanol-acetic acid. Most of the specimens were embedded
in ester wax (Steedman, 1961), cut at 2 pu and stained by the Feulgen method.
From some specimens squashes of retinal tissue were prepared by the method
described in a previous publication (Denham, 1966). Autoradiographs were
made from the preparations by coating the slides with Ilford Nuclear Research
Emulsion K 5 diluted 3:1 with distilled water at 40 °C. The slides were dried in
air and stored at 4 °C in light-tight boxes containing silica gel. The duration of
exposure varied from 30 days to 4 months depending on the amount of thymidine used. Autoradiographs were developed in D 19 b for 4min at 18 °C and
subsequently fixed in Johnson's Fixol diluted 1:10 for 10 min.
In one group of animals metaphase cells were arrested with Colcemid.
Approximately 0-007 mg/g of body weight was injected with a micrometer
syringe. This concentration effectively arrested metaphases for 4 h; higher concentrations proved lethal within 3 h.
Cells from some adult rat eyes were prepared for Feulgen spectrophotometry
in the following manner. After fixation in ethanol-acetic acid the eyes were
rehydrated and the neural retina dissected free from the pigment epithelium
and connective tissue. The retina was stained by the Feulgen reaction and
subsequently homogenized in water with a Waring blender for 20 s. This procedure effectively separated the tissue into cells; the nuclei were preserved intact
55
Rat neural retina
and, although the cell cytoplasm was obviously damaged, very little nuclear
debris was seen in the final preparations. The homogenate was transferred to a
centrifuge tube, dehydrated with alcohol and cleared with xylene, each solution
being removed as a supernatant after gentle centrifugation. The cells were
finally mounted in oil of the same refractive index on a microscope slide.
Subsequent photography (to facilitate localization) of the cells and spectrophotometric readings of the Feulgen density of their nuclei were kindly carried
out by Dr Smith and Dr Dendy of the Department of Radiotherapeutics,
University of Cambridge. The apparatus used for the spectrophotometry was
that designed by Mendelsohn (1958).
Counting methods
Only sections passing through the optic nerve were used for analysis, and
counts were made solely in the areas around the optic papilla, i.e. the 'central
third' of the retina (see Text-fig. 1). This was done to ensure that analyses were
Neural retina
Central third
Optic nerve
Pigment epithelium
ONL
Text-fig. 1. Diagrammatic representation of the eye to show the position of the
'central third' of the area of the neural retina and the cell layers of the mature
retina. OGC, optic ganglion cells; INL, inner nuclear layer; ONL, outer nuclear
layer.
confined to areas of the same developmental stage. Optic ganglion cells were
never included in the cell counts, because in 2-day-old animals, these cells were
always differentiated and well separated from the rest of the retina by an inner
fibre layer (Plate 1, fig. A). A strip graticule 1 mm wide orientated at right
angles to the retinal circumference was used to measure samples of the total
retinal cell population.
Since the maximum number of grains found over fully differentiated nuclei
56
S. DENHAM
in autoradiographs was never above two, cells were scored as 'labelled' if 5 or
more grains covered or were immediately adjacent to the nucleus. The duration
of the cell generation cycle was determined by the method of Sherman &
Quastler (1959), by recording the incidence of labelled mitoses at various intervals after administration of tritiated thymidine. In one experiment the distribution of grains per nucleus was assessed in 2-day animals killed at 1 h, 2 h,
24 h and 10 days after injection of labelled thymidine. A line graticule was
moved progressively through a selected part of the specimen and the grain level
of all labelled cells in the path of the graticule was recorded.
EXPERIMENTS AND RESULTS
At 2 days the neural retina of the rat in section consists mainly of a wide
band of cells whose nuclei appear undifferentiated; this band is bordered on its
inner margin by a layer of well-differentiated optic ganglion cells from which it
is separated by a clearly denned inner fibre layer (Plate 1, fig. A). The nuclei of
these undifferentiated cells are large, ellipsoidal and stain densely (Plate 1,
fig. B) but at the extreme inner border of this layer there are a few nuclei (about
10 % of the total outer undifferentiated layer) which show signs of early differentiation in that their shape is more nearly spherical and they stain less
intensely than their neighbours. These innermost cells are the first cells of the
future inner nuclear layer. Mitoses, with a few rare exceptions, occur only along
the outer perimeter of the 2-day neural retina, immediately beneath the external
limiting membrane. By the time a rat is 5 days old, in the central area of the
retina, the undifferentiated cell layer has developed into the inner and outer
nuclear layers of the adult eye (Plate 1, fig. C), and the nuclei have acquired a
characteristic morphology depending on the layer to which they belong. The
areas of the retina around the optic papilla are always more advanced in
development than the ciliary regions and differentiation progresses from the
centre towards the iris. Although cell proliferation has largely ceased in the
central retina of the 5-day-old rat, the ciliary regions are still undifferentiated
and have abundant mitotic activity.
When 2-day-old rats were injected with tritiated thymidine and killed an hour
afterwards, autoradiographs revealed that labelled cells were situated in a
discrete band in the deeper layers of the undifferentiated cell layer (Plate 1,
fig. D). No labelled cells were found in the differentiating inner nuclear layer or
among the optic ganglion cells, but some nuclei of endothelial cells adjacent to
the optic ganglion cells were labelled. When animals were killed at longer
intervals after thymidine injection, autoradiographs of the retina showed that
labelled nuclei migrated to the outer, cell-free edge, before division and that the
daughter nuclei moved inwards after mitosis.
The percentage of mitotic figures labelled at different intervals after injection
of tritiated thymidine into 2-day-old animals was recorded graphically and the
J. Embryo/, exp. Morph., Vol. 18, Part 1
m.
PLATE 1
A
*M£frli
*j^^^F
(ii.
D
Fig. A. Histological section through the 2-day-old rat retina near the optic papilla, o, Optic
ganglion cell layer; /./., inner fibre layer; /.//., inner edge of future inner nuclear layer; m;
mitotic figure adjacent to the outer limiting membrane, x 254.
Fig. B. Squash preparation of a 2-day retina from an animal which was killed 1 h after
injection with tritiated thymidine, showing the nuclear morphology of the undifferentiated
cells. x815.
Fig. C. Histological section through the 5-day-old rat retina, o, Optic ganglion cell layer;
/./., inner fibre layer; /./?., inner nuclear layer; o.n., outer nuclear layer; o.f., outer fibre layer.
x254.
Fig. D. Section through the retina of a 2-day-old rat 2 h after injection of tritiated thymidine.
o, Optic ganglion cell layer; /'./., inner fibre layer; /./?., future inner nuclear layer; /., labelled
nuclei; in, mitotic figure, x 254.
S. DENHAM
facing p. 56
Rat neural retina
57
duration of the retinal cell generation cycle determined from the graph after
the method of Sherman & Quastler (1959). The result is shown in Text-fig. 2
and summarized in Table 1. The experiment was performed twice and the
curve in Text-fig. 2 is drawn through the results of the second experiment (solid
circles), in which 3 /^Ci. of tritium per g of animal was administered. The incomplete results of an initial experiment (open circles) in which 1 j^Ci./g was used are
superimposed to demonstrate that increasing the dose of radioactivity did not
appear to affect the timing of the generation cycle.
100
90
80
|
70
T3
60
1 50
JS
fp 40
co
I
30
20
10
2 4 6 8 1012141618 2022242628303234 36384042 444648505254 565860
Time (h)
Text-fig. 2. Percentage labelled mitotic figures vs. time in hours after injection of
tritiated thymidine in the 2-day neural retina. • , H3Tdr 3/tCi./g; O, H3Tdr, l/tCi./g.
Table 1. Duration of cell cycle phases in 2-day rat retina
Phase...
Duration (h)
Total generation time (h)
S
G2
M
Gl
12-5
1-5
0-8
13
28
The first ascending limb of the curve corresponds to the first wave of labelled
cells passing through mitosis. Almost 100 % of the mitotic figures become
labelled and remain so for a period corresponding to the duration of the synthetic phase; as cells which were in G1 when thymidine was available enter
mitosis, the index for labelled mitotic cells falls. The second ascending limb of
the curve represents the cohort of labelled cells entering mitosis for the second
time. The interval between the 50 % intercepts on the first and second ascending
58
S. DENHAM
limbs of the curve corresponds to the duration (28 h) of the total cycle. The time
from administration of the label (at 0 h) to the 50 % intercept on the first limb of
the curve represents G2 plus half the average duration of M, i.e. 2 h. No difference was found between the time at which 50 % of all clearly recognizable
mitoses, and that at which 50 % of metaphases only, were first labelled; subsequently, therefore, all mitotic stages were counted. The duration of S is represented by the distance between the corresponding 50 % points on the limbs of
the first peak of the curve. If G2 + i M and S are substracted from the duration
of the total cycle, the duration, of Gx + ^M is obtained, i.e. 13^ h.
500
r
2
Time (h)
3
4
Text-fig. 3. Accumulated metaphases after administration of Colcemid to 2-day-old rats.
The duration of the mitotic process itself was determined from separate
experiments. Two-day-old rats were treated with Colcemid; two animals were
killed immediately after treatment and two at hourly intervals thereafter up to
4 h. This procedure was repeated twice and the accumulated metaphase indices
from 0 to 4 h for all three experiments were recorded. The result is shown in
Text-fig. 3. From the pooled results a mean figure of 1-3 for the number of
mitoses per hour was obtained and the average mitotic duration, therefore, was
1/1-3 or 0-8 h. For the purpose of determining the lengths Gx and G2 the value
obtained for the duration of M was approximated to 1 h.
Rat neural retina
59
Cell generation cycle in the 5-day-old rat
In the 5-day-old rat, cell proliferation in the central areas of the neural retina
has largely ceased and the mitotic index is reduced to 0-08 %. Cell division still
persists in the ciliary retinal areas, however, and it Seemed interesting to compare the generation cycle of cells in the central retina with similar cells that were
probably nearer the end of their proliferative life. The generation cycle of cells
100
90
80
70
60
JJ 50
<L>
40
30
20
10
4 6 8 10121416182022242628303234 36384042444648
Time (h)
Text-fig. 4. Percentage labelled mitotic figures vs. time in 5-day-old rat neural
retina, to show the duration of the cell generation cycle. • , H3Tdr, 3/tCi/g; O,
H 3 Tdr, 1 /tCi/g.
in the ciliary retinal areas of 5-day-old rats was measured by the same method
as that used for younger animals, and the resultant curve is shown in Text-fig. 4.
The results obtained for 5-day animals are less complete than those for two-day
rats but they nevertheless show there are no major differences in the duration
of the cell cycle and its phases between 2 and 5 days.
The phase during which cells leave the generation cycle
The following experiments were made to test the assumption that proliferative
cells usually leave the generation cycle from the Gx phase prior to entering a
transitional or differentiating population.
(1) Comparisons of the number of grains per labelled cell were made (a)
between 2-day rats killed 1 h and 24 h after injection of tritiated thymidine, and
(b) between 2-day animals killed 2 h and 10 days after labelling. Experiment
(a) allowed sufficient time for all labelled cells to have passed through mitosis
60
S. DENHAM
once in animals killed 24 h after labelling. In experiment (b) it was expected that
the grain distribution per cell in 12-day-old animals would be representative of
that in the fully adult retina, as by this time cell division in the central retina
would have long ceased. Grain distributions per labelled cell for the rats in each
experiment are shown as histograms in Text-figs. 5 and 6. In both experiments,
if a significant proportion of the cell population had synthesized DNA without
subsequent division, at 24 h and 10 days a secondary peak corresponding to
the peak level found in animals killed at 1 and 2 h respectively would be detectable. In neither experiment (a) nor (b) is such a secondary peak noticeable.
30 r
J2
8
25
20
20
15
CD
60
10
CO
I
5-9
10
I
15-19
25-29
35-39
4
6
8 10 12 14 16 18 20
5 7 9 11 13 15 17 19
Number of grains
Fig. 6
10-14
20-24
30-34
Number of grains
Fig. 5
Text-fig. 5. Percentage distribution of grains in labelled cells of the neural retina
1 h (•) and 24 h (D) after administration of tritiated thymidine to 2-day-old rats.
Text-fig. 6. Percentage distribution of grains in labelled cells of the neural retina
2 h (•) and 10 days (d) after administration of tritiated thymidine to 2-day-old rats.
(2) If any neural retinal cells leave the generation cycle after DNA synthesis
but before entering mitosis, they should remain in the adult population as cells
having twice the normal diploid complement of DNA. Fifty cells of the outer
nuclear layer and 50 of the inner nuclear layer were stained by the Feulgen
method and assessed for their DNA content by a spectrophotometric method.
One of these nuclei had a DNA value which fell outside the range of the normal
distribution curve obtained for the rest of the nuclei, but on closer inspection
it appeared that this 'nucleus' was actually two superimposed nuclei.
In these experiments no significant proportion of cells having twice the normal
diploid DNA content could be found, and thus it seems likely that neural retinal
cells leave the generation cycle from Gx prior to differentiation.
The size of the proliferative population at 2-days and
its manner of growth
Since the S phase accounts for about 45 % of the total duration of the cycle,
the percentage of labelled nuclei should also be approximately 45 % if all the
Rat neural retina
61
cells of the retina were in the proliferative compartment. (If all the cells were
proliferative the number in S would only approximate to 45 % as formulae for
exponential growth would apply, see Smith & Dendy, 1962). Since only about
15 % of the cells are labelled after a single injection, the proliferative compartment accounts for only a third of the cells in the neural retina at this stage.
15
1-5
8 io
10 8
•d
1
bQ
1 5
20
0-5
48
48+28
= 76
76+28
= 104
I
104+28
=132
Hours after birth
Text-fig. 7. Decrease in labelling and mitotic indices in the neural rat retina 2-5
days after birth. • - - - • , Theoretical decrease in labelling index if growth is linear;
A---A, theoretical decrease in labelling index if growth represents only two-thirds
of the linear increase; • — • , measured decrease in labelling index; O—O,
measured decrease in mitotic index.
If the size of the neural retinal population increases linearly, i.e. if half the
daughter cells produced by mitosis join the differentiating population and half
proceed through another generation cycle so that the population grows by a
constant amount at each cycle, then predictions for the theoretical decrease in
labelling index can be made for each cycle covering the period from 2 to 5 days.
The labelling index in animals aged 48 h (2 days), 76 (48 + 28) h and 104 (87 + 28)
h hah0 an hour after injection of tritiated thymidine, was determined from
squash preparations. The labelling index in 5-day (120 h) old rats 2 h after
thymidine administration was determined from sections, as retinas of this age
were too fibrous to make good squash preparations. In Text-fig. 7 the observed
changes in labelling index are compared with the theoretical decrease in labelling
index to be expected if the total retinal growth were linear from 2 to 5 days.
Text-fig. 7 also shows a theoretical curve for the decrease in labelling index if
two-thirds of the total number of daughter cells produced each cycle leave the
62
S. DENHAM
proliferative population, i.e. if growth is less than linear from 2 to 5 days. The
observed decreases in mitotic index in animals killed at 48, 72 (3 days) and 120
(5 days) hours are also given to compare the reduction of this index with that of
the labelling index.
It can be seen from the graph that growth appears to be less than linear from
2-5 days onwards, i.e. more than 50 % of the daughter cells leave the proliferative population and the absolute number of proliferative cells decreases at each
cycle. Mitotic indices fall in a similar manner to the observed labelling indices,
although the mitotic index at 3 days has decreased less than the labelling index.
Unfortunately, it is impracticable to trace the labelling index back to an earlier
stage of development than 48 h after birth, as problems of interpretation would
be presented by perinatal depression of mitotic activity. Thus, it would be
difficult to assess whether growth of the retina might still be linear at 2 days or
whether, as seems most likely, differentiation is already proceeding at a faster
rate than cell production. While for the period from 2 to 4 days the theoretical
curve based on two-thirds linear growth is close to the observed figures, by the
5th day the proliferative compartment of the retina has shrunk at a faster rate
than two-thirds which is confirmed by morphological observations and the
development of the outer fibre layer (Plate 1, fig. C).
DISCUSSION
The undifferentiated cell population present in the immature neural retina
(called 'matrix' cells by Fujita, 1962) develop into two functionally and morphologically different cell types: the inner nuclear layer, composed mainly of bipolar
cells with additional horizontal and amacrine cells, and the outer nuclear layer
containing the photoreceptor cells. There are, however, reasons for believing
that the undifferentiated cells behave as a homogenous population. The curve
obtained for the retinal generation cycle at 2 days is very clearly defined for the
first wave of labelled cells passing through mitosis, and if two cell types possessing
different patterns of timing for the generation cycle were involved, such definition
would be difficult to obtain. Furthermore, Sidman (1961) demonstrated that
during the development of the mouse retina after mitosis labelled cells may
return to either the inner or the outer nuclear layer before differentiation.
The figure of 28 h obtained for the duration of the generation cycle falls well
within the range of cycle lengths found for other types of cells. The shortest
generation cycles occur in tissues undergoing the most rapid proliferation, e.g.
in the early stages of embryogenesis. In the 1-day-old chick embryo, cells of the
neural retina have a generation time of 5 h (Fujita, 1962) but at 6 days of age,
the cycle has lengthened to 10 h. In the same animals, cells in the basal plate of
the neural tube have an estimated generation time of 2-5 days (Fujita, 1962).
Neural tube cells of 10-day-old mouse embryos were found to have a life cycle
of 8-5 h duration. The shortest generation times so far encountered in adult
Rat neural retina
63
mammalian tissues seem to be about 11-12 h (Cattaneo, Quastler & Sherman,
1961; Lesher, Fry & Kohn, 1961a; Defendi & Manson, 1963) and the longest,
in the epidermis of the mouse ear, about 2-3 weeks (Sherman, Quastler &
Wimber, 1961). The generation cycle of the 2-day old rat retina is long as compared to that observed in other embryonic neural tissues (Fujita, 1962; Kauffman,
1966), but the latter studies were made on much younger animals, At 2 days the
rat neutral retina is approaching the end of its proliferative phase and there is
evidence that the cell generation cycle lengthens with age for both adult
(Lesher, Fry & Kohn, 1961 b) and embryonic (Fujita, 1962; Graham & Morgan,
1966) tissues.
It has been suggested (Defendi & Manson, 1963) that the duration of the G2
and S phases remain roughly constant for all mammalian cell types and that
differences in cycle timing are primarily due to differences in the length of Gx;
in some tissues this has been verified (Cattaneo, Quastler & Sherman, 1961).
The figure normally accepted as being the average duration of S is, however,
about 6-8 h (Cameron, 1964; Defendi & Manson, 1963), about half the value
for S obtained in the present experiments. There is some indication that the S
phase may lengthen with age in the embryonic neural tube of the mouse (Kauffman, 1966). Cameron, working with chicks, has shown that the duration of S
in a particular tissue may be related to its temperature (Cameron, 1964).
Although the neural retina, being a peripheral organ, may suffer a decrease in
temperature after birth, it is difficult to believe that this would be sufficient to
increase S by a factor of two. Cameron & Cleffman (1964), for example, found
that in several cell populations in the chick a fall in temperature of approximately 3 °C was needed to prolong S from 5-6 h to 6-9-7 h. It seems likely that
age, in the sense of an advance towards differentiation, is responsible for the long
duration of S in the neonatal rat retina. For early amphibian development
Graham & Morgan (1966) have shown a progressive increase with age in all
phases of the cycle except that of M. Apparently, some cells with a lengthy
cycle may spend much longer in the S phase than most other cells; for example,
in the epidermis of the adult mouse ear, S occupies 30 h out of a total cycle time
of 2-3 weeks (Sherman et ah 1961) and some tumour cells with generation times
greater than 24 h have an S phase of 13 h or more.
The G2 of 1-5 h and an M of about 1 h measured in the present experiments
are comparable with values obtained by other workers for developing neural
tissue. Thus Fujita (1962) found G2 to be about 2 h and M approximately 1 h
in various parts of the neural tube of the 6-day chick embryo, and Kauffman
(1966) showed that G2 was just less than, and M just more than an hour in the
neural tube of the 10-day mouse embryo.
The author found no evidence for the presence of tetraploid nuclei in the
neural retina of the adult rat, nor for any significant number of cells remaining
in G2 in the early post-natal stages of development. Although there is some
evidence that in one tissue, cells enter an abnormally long G2 after synthesis
64
S. DENHAM
(Gelfant, 1962), the occurrence of a metabolic synthesis of DNA (Pelc, 1963)
has often been evoked to explain the existence of labelled cells that do not
divide after synthesis and the appearance of label over differentiated cells
(Lasnitzki & Pelc, 1957; Owen, 1963). Thus the suggestion of Hicks et al. (1961)
that retinal cells might replicate their chromosome content and then remain
permanently in G2 has not been confirmed. This does not preclude the possibility, however, that a few cells (too small a number to register a peak on the
histograms) might remain temporarily in a G2-associated Go as postulated by
Quastler (1963). These results appear to confirm the assertion of Fujita (1964)
that no DNA synthesis occurs in the central nervous system once the cells have
differentiated.
Only about 10 % of the cells in the neural retina of the 2-day-old rat appear
to be morphologically differentiated. Thus over 60 % of the population, though
morphologically similar to the proliferative cells, do not participate in DNA
synthesis or division. It would be interesting to know whether these cells have
irreversibly left the proliferative pool or whether they have entered a Go or
transitional phase from which they could return in case of injury to the immature
retina, e.g. after damage by X-radiation, and be capable of reproduction in the
repair of the injured tissue.
SUMMARY
1. An autoradiographic method was used to study cell proliferation in the
neural retina of the neonatal rat. The cell generation cycle in the retina of the
2-day-old animal was determined by the percentage of labelled mitosis after
administration of tritiated thymidine. The cycle had a total duration of 28 h,
S = 12-5, G1 = 13, G2 = 1-5 and M = 0-8 h.
2. The cell generation cycle of 5-day-old rat retina was almost the same as
that in younger animals.
3. The distribution of grains in labelled cells from rats killed 24 h and 10 days
after thymidine injection, and Feulgen spectrophotometry of nuclei from adult
rats, failed to show any significantly prolonged or permanent stay in G2 after
DNA synthesis.
4. Labelling indices were measured at 48, 48 !-28, 76 + 28 h and at 5 days.
From the degree of decrease in labelling index from 2-5 days it was concluded
that growth over this period was about two-thirds linear and that only one-third
of the cell population at 2 days might still be in the proliferative compartment.
RESUME
Etude de la proliferation cellulaire de la re'tine nerveuse chez
le rat de deux jours
1. On a utilise une methode autoradiographique pour etudier la proliferation
cellulaire dans la retine nerveuse du rat nouveaune. On a determine le cycle d'une
Rat neural retina
65
generation cellulaire dans la retine de l'animal de 2 jours, a l'aide du pourcentage de mitoses marquees apres l'administration de thymidine tritiee. Le
cycle a une duree totale de 28 h: S = 12h30, G l = 13 h, G2 = 1 h 30,
M = 50 min.
2. Le cycle de generation chez des rats de 5 jours etait presque le meme que
celui d'animaux plus jeunes.
3. La repartition des grains dans les cellules marquees de rats tues 24 h et
10 jours apres Tinjection de thymidine et la spectrophotometrie de noyaux de
rats adultes, colores au Feulgen, n'ont pas montre de prolongation significative
ou de permanence de la phase G2 apres la synthese d'ADN.
4. Les indices de marquage ont ete mesures a 48, 48 + 28, 76 + 28 h et a 5
jours. Du degre de diminution de l'indice de marquage du 2eme au 5eme jour
on a conclu que la croissance au cours de cette periode a ete lineaire pour
environ les 2/3 et qu'un tiers seulement de la population cellulaire au 2eme jour
pourrait encore se trouver dans la fraction proliferante.
The author is indebted, to Dr A. Gliicksmann for supervising this research and for assisting
with the manuscript. I am also grateful to Dame Honor Fell for revising the manuscript and
to Professor Bullough for providing laboratory facilities. This research was given financial
support by the British Empire Cancer Campaign for Research and, in part, by the Wellcome
Trust.
REFERENCES
M., ROGERS, A. W. & EAYRS, J. T. (1964). Pattern of cell migration during cortical
histogenesis. Nature, Lond. 203, 591-3.
CAMERON, I. L. (1964). Is the duration of DNA synthesis in somatic cells of mammals and
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{Manuscript received 31 January 1967)