/. Embryol. exp. Morph. Vol. 34, 2, pp. 497-5JO, 1975
Printed in Great Britain
497
Cell cycle parameters in dedifferentiating
iris epithelial cells
ByTUNEO YAMADA, 1 MARION E. ROESEL 2 AND
JOHN J. BEAUCHAMP 3
From the Biology Division, Oak Ridge National Laboratory, Tennessee,
Institut Suisse de Recherches Expe'rimentales sur le Cancer, Lausanne and
the Mathematics and Statistics Research Department, Union Carbide
Corporation, Nuclear Division, Oak Ridge, Tennessee
SUMMARY
After lentectomy of the adult newt eye, non-dividing iris epithelial cells re-enter the cell
cycle. Some of the iris epithelial cells become completely depigmented while they are in the
cell cycle, and then differentiate into lens cells. The remaining iris epithelial cells become
partially depigmented in the induced cell cycle, but resynthesize melanosomes and recover
the normal state of iris epithelial cells. The two groups of cells are spatially separated within
the iris epithelium. The cell cycle parameters of both groups of iris epithelial cells were estimated by a mathematical procedure on a computerized programme from the percentage of
labelled mitotic cells as a function of time after peritoneal injection of [3H]methyl-thymidine
on day 6 after lentectomy. The total cell cycle time was found significantly shorter in the cell
population with complete depigmentation as compared with that with partial depigmentation.
Based on these results the possible role of differential cell cycle time in the control of dedifferentiation was discussed. Grafting of unlabelled iris into the optic cavity of host animals
injected 6 h beforehand with [3H]methyl-thymidine, followed by a study of radioactivity of
iris epithelial cells of the graft demonstrated incorporation at a low level during the whole
period of the experiment in which the cell cycle parameters were estimated. The data used
for the estimation were corrected for the delayed incorporation.
INTRODUCTION
Lentectomy of the adult newt eye is followed by regeneration of a functional
lens. The classical notion that this lens is derived from the iris epithelium (IE)
was supported by a considerable amount of circumstantial evidence (for review
Yamada, 1967). Recent tissue culture experiments (Connelly, Ortiz & Yamada,
1973; Yamada, Reese & McDevitt, 1973; Yamada & McDevitt, 1974) have
provided a direct proof for the notion. This implies that upon lentectomy, fully
differentiated non-dividing adult IE cells (Eguchi, 1963; Karasaki, 1964;
Yamada & Roesel, 1969, 1971; Dumont & Yamada, 1972) reverse their
1
Author's address: Institut Suisse de Recherches Experimentales sur le Cancer, Ch 1011
Lausanne, Switzerland.
2
Author's address: 9235 Happy Lane, Oak Ridge, Tennessee 37830, U.S.A.
3
Author's address: Mathematics and Statistics Research Department, Computer Sciences
Division, Union Carbide Corporation Nuclear Division, Oak Ridge, Tennessee 37830, U.S.A.
498
T. YAMADA, M. E. ROESEL AND J. J. BEAUCHAMP
established state of differentiation and engage in a developmental pathway which
they do not follow during their ontogenesis. The whole sequence of events, which
is induced by lentectomy in the IE cells can be divided into three processes: induction of cell replication, dedifferentiation, and redifferentiation (Yamada, 1972).
The earliest cellular change observed after lentecomy in IE cells is activation
of the nucleoli (Eguchi, 1963; Karasaki, 1964; Dumont, Yamada, & Cone,
1970) which is associated with an enhancement of ribosomal RNA synthesis
(Reese, Puccia & Yamada, 1969; Reese, 1973; Jauker & Yamada, 1973).
Subsequently the cells enter the first induced DNA synthetic phase (Eisenberg
& Yamada, 1966; Reyer, 1971; Yamada & Roesel, 1969; Eguchi & Shingai,
1971) which is followed by the first wave of mitoses around 4 to 5 days after
lentectomy (Yamada & Roesel, 1971). While the IE cells are in the cell cycle,
the population of melanosomes is progressively eliminated - a process traditionally called depigmentation. After depigmentation has been completed, the cells
form a lens vesicle and withdraw from the cell cycle as they differentiate into
lens fiber cells (Eisenberg & Yamada, 1966; Reyer, 1971; Eguchi & Shingai,
1971). Since completion of depigmentation in IE cells coincides in time and
location with the appearance of competence for lens formation, one can argue
that during the depigmentation phase the cells not only lose their overt differentiation but also become disengaged from their original commitment. Hence the
phase of depigmentation can be identified with the phase of dedifferentiation.
Depigmentation of IE cells appears to be caused mainly by discharge of
melanosomes from the cell body (Wolff, 1895; Eguchi, 1963). The most important mode of discharge is separation from the cell of a package of melanosomes
bound by membrane (Dumont & Yamada, 1972, unpublished). During the
depigmentation phase which lasts for 6 days or more, the surface of IE cells
sends out extensive projections, and the IE becomes infiltrated by macrophages
and neutrophils which take up the discharged materials (Eguchi, 1963; Yamada
& Dumont, 1972). On the other hand, the absence of premelanosomes in IE
cells undergoing active replication induced by lentectomy implies that synthesis
of melanosomes is not coupled with cell replication. Hence during depigmentation, cell replication should also contribute to reduction in the melanosome
number per cell by dilution.
One more aspect needed as background information in the present paper is
related to the fact that cell replication is induced all over the iris ring by lentectomy, but that only some of those proliferating IE cells become transformed
into the lens cells. The discussion made in the preceding paragraph concerns
this latter population of IE cells. The remaining IE cells induced in replication
go through a process of partial depigmentation, resynthesize melanosomes, and
resume differentiation as IE cells as they withdraw from the cell cycle (Eisenberg
& Yamada, 1966; Reyer, 1971; Yamada & Roesel, 1971; Eguchi & Shingai,
1971). Thus alternative pathways are open to IE cells by lentectomy: one
involves definitive dedifferentiation and leads to lens cell differentiation, and
Dedifferentiating iris epithelial cells
499
the other involves transient dedifferentiation and leads back to the normal state
of IE cells. So far as lens regeneration in situ is concerned, the choice of pathways
is determined by the location of cells within the IE. This also applies to repeated
lens regeneration induced by repeated lentectomy.
In the efforts to elucidate the nature of the dedifferentiation process by various
approaches (Zalik & Scott, 1972, 1973; Ortiz, Yamada & Hsie, 1973; IdoyagaVargas & Yamada, 1974), information on the cell cycle parameters of IE cells
during lens regeneration in situ is indispensable. The main part of this paper
concerns estimation of the duration of cell cycle phases of IE cells involved in
the alternative pathways of dedifferentiation during lens regeneration in situ. The
autoradiographic data on the percentage of labelled mitoses as a function of
time after peritoneal injection of tritiated thymidine into the adult newt 6 days
after lentectomy were used for the basis of computation. At this time the IE
cells begin active depigmentation and the strict synchronization in the DNA
synthesis, which is characteristic for the preceding period but inconvenient for
the present analysis, has been lost.
In order to estimate the average times spent in the three major cell cycle phases
(presynthetic, DNA synthetic, and post-synthetic), we assumed that the time
spent in these three phases follows a truncated normal distribution. An expression
has been derived to describe p(t), which is the percentage labelled cells in the
mitotic phase at successive times t. Since p{t) is a non-linear function of six
unknown parameters (averages and variances for the distributions of times
spent in the three phases) and the variance of the observed values of p(t)
depends upon t, an iterative non-linear estimation procedure was used to obtain
the weighted least-square estimates of these quantities.
In another group of experiments grafting of iris tissue and autoradiography
were combined to test the possible persistence of cell labelling activity in the
present system. A low level of cell labelling activity in the optic environment was
demonstrated after the end of the primary labelling period due to injection
of labelled precursor. The percentage of labelled mitotic cells (MC) used to
estimate cell cycle phases were corrected for the delayed incorporation.
MATERIAL AND METHODS
Biological material. Adult newts (Notophthalmus (Triturus) viridescens)
collected in East Tennessee (Lee's Newt Farm, Oak Ridge) and kept in spring
water in laboratory tanks at 21-22 °C under continuous illumination were
exclusively used.
Lentectomy. The animals were anesthetized with Tricaine (Sigma), a paper
plug was inserted into the buccal cavity, and a horizontal incision was made in
the cornea. The lens was removed bilaterally through the incision by a gentle
push on the dorsal and ventral areas of the cornea.
Cell labelling and autoradiography. In experiment I the lentectomized animals
500
T. YAMADA, M. E. ROESEL AND J. J. BEAUCHAMP
were injected intraperitoneally with 3 /*Ci/g body weight of [3H]methyl-thymidine
(specific activity 14 Ci/mmole, Schwarz BioResearch) 6 days after lentectomy. The animals were sacrificed at 4 or 8 h intervals during the period
of 4-112 h after injection. Subsequent to fixation of the head in Carnoy, the
eyeballs were separated, embedded in paraffin, and sectioned serially at 5 [ivs\
thickness. The orientation of the sections was sagittal to the eye as an independent bilateral body. The mounted sections were treated with 10 % hydrogen
peroxide for 22 h to achieve partial bleaching of melanin. The possible effects
of the bleaching procedure on autoradiographic counts was checked and found
insignificant. The sections were covered with NTB 3 emulsion (Kodak), exposed
for 2 weeks at 4 °C, and developed with D 11 developer (Kodak) for 3 min at
20 °C. Mayer's hemalum was used to stain chromatin.
DR
LFA
VR
d
Non-LFA
e
Fig. 1. Compartments of iris epithelium used in Expt. I. (A, B) diagrams of the
eye with the dorsal side up, indicating location and extent of the four compartments.
The antero-posterior organization of the eye and the presence of the iris stroma
are not taken into account. (C) Dorsal iris epithelium composed of LFA and
dorsal non-LFA. d-d and e-e indicate the levels of the saggital section of IE
shown in (D) and (E) respectively. In (D) the dorsal one-half of the inner lamina and
the distal one-fourth of the outer lamina of IE are indicated as LFA. In (E) the
dorsal one third of the inner lamina alone belongs to LFA. Diagrams (B-D) are based
on autoradiographic tracing of lens-forming cells (Eisenberg & Yamada, 1966;
Reyer, 1971; Eguchi & Shingai, 1971), and are comparable to the embryological fate
maps. But in the absence of data from localized vital staining and genetic mosaics
the accuracy of those diagrams is less than that of the established fate-maps.
Compartments of IE. In obtaining the percentage of labelled MC in Expt. I,
the IE was divided into topographical compartments which are designated as
dorsal region (DR), ventral region (VR), lens-forming area (LFA) and nonlens-forming area (non-LFA) as explained in Fig. 1. LFA is a part of DR,
Dedifferentiating iris epithelial cells
501
while non-LFA comprises the remaining part of DR and the whole VR. The
cells which participate in lens cell differentiation after complete depigmentation
are all located in LFA, while the cells of non-LFA retain IE specificity after
incomplete depigmentation.
Methods for counting labelled and unlabelled MC. In Expt. I, the complete
set of serial tissue sections was scanned under a microscope, and in each
compartment of IE, the total numbers of labelled and unlabelled MC were
recorded. One mitotic IE cell is sectioned into 2-4 consecutive slices. Since in
the sectioned IE the nuclei are widely dispersed, and the number of IE cells
per tissue section is limited, it was possible to follow a single mitotic cell through
the serial tissue sections. The grain counts of single MC were obtained by addition of silver grains over all slices of the cell. Since Expt. II demonstrated
progressive low-level labelling of cells in cell cycle during the time interval
employed in Expt. I, it became necessary to consider the delayed labelling in
our method of distinguishing labelled and unlabelled MC. This was done by
using the following set of minimum grain counts per MC in determination of
labelled MC: 8 grains for 4-20 h series; 10 grains for 24-44 h series. 11 grains
for 48-68 h series; 12 grains for 72-92 h series; 13 grains for 96-112 h series.
These counts are based on the data of Expt. II on delayed incorporation in the
optic cavity, which demonstrated that the incorporation is a function of the
time interval during which the cells are exposed to the environment.
Estimation of cell cycle parameters. For convenience, we use the following
notation for the cell cycle phases: (1) presynthetic (Gl); (2) DNA synthetic (S);
(3) postsynthetic (G2); and (4) mitotic (M). The phase duration of M is very
short, so we split it between Gl and G2 in our modelling procedure. Let Xly X2
and X3 represent the time a cell spends in S, G2, and Gl respectively, andp{t)
designate the probability a cell in mitosis at time t shows labelling. It has been
shown (Okumura, Onozawa, Morita & Matsuzawa, 1973) that the expression
for p(t) involves the convulution of the distributions for Xx, X2, X3. In our
modelling effort truncated normal distributions, which do not allow negative
values of our random variables as normal distributions, would have been used
for Xlt X2 and X3.
The observed data, p(t), are given as the proportion of labelled cells in M at
successive times /, that is
where 1 -> (t) is the observed number of labelled MC at time / and N(t) is
the observed number of MC at time t. The expression for p(t) involves six
unknown parameters (three unknown mean or average times spent in the phases
and three unknown variances) of the distributions for Xlt X2 and X3.
The weighted-least-squares estimates of these parameters were obtained by
minimizing
^
alH
502
T. YAMADA, M. E. ROESEL AND J. J. BEAUCHAMP
where w(t) is the weight associated with the observation p(t) at time /. In this
study w(t) was chosen equal to N(t)![p(t)(\—/p(t))] under the assumption that
the observed values p(t) followed a binomial distribution. Since p(t) is a nonlinear function of the six unknown parameters, an iterative estimation technique
was used to obtain the estimates in Table 1. The computer program used was
the program N0NLS2 written by Wesley & Watts (1970) based on the non-linear
estimation technique of Marquardt (1963).
toti cells
8
o
E
100 -
f«MMI MM
•
B. VR
80•
6040-
,
•
* . •
•
—
Label
• •
•
•
* ' *
•
20 :
t
T i l U '
0
100 -
8
,
u
40 t 60 • 80 h00112 h
8d
9d
10 d
— .
80-
8
C. LFA
• •
1
40-
bell
•
20-
• .
•••
• ••
3
• •
•
•
• •
• :
•
• •
• • •
•
•
i'
20'
7d
0
20*
7d
100-
i .
•• • •
60-
O
-a
u
20t
7d
40 ' 60 f 80 '100112
8d
9d
10 d
40 • 60 • 80 h00H2 h
8d
9d
10 d
D. Non-LFA
I M M H I *
806040-
•
•
•
•
#
•
•
•
r
•
•
•••
••
20-
•:
*
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-
• "
i
i
.
1
:
20'
7d
i
l
i
40 ' 60 ' 80
8d
9d
M00112 h
lOd
Fig. 2. The percentage of labelled mitotic cells of the four compartments of iris epithelium as a function of time after injection. On the abscissa, h signifies hours
after injection, and d days after lentectomy.
Methods used in Experiment II. A group of donor animals was lentectomized.
Eighteen days later another group of animals which was to serve as hosts was
injected peritoneally with 3 /*Ci/g body weight with [3H]methyl-thymidine, and
lentectomized through a U-shaped incision in the cornea. The shape of the
incision was selected to facilitate retention of the graft subsequently implanted
into the optic cavity. Six h after injection the regenerating lenses were removed
from the donors and grafted into the host optic cavity through the incision.
The hosts were kept alive for 24 or 120 h at 21-22 °C Then their heads were
Dedifferentiating iris epithelial cells
503
fixed in the Carnoy fixative, and the eyes were embedded in paraffin and
sectioned serially into sections 5 /im thick. The mounted sections were processed
for autoradiography as described above.
RESULTS
Experiment I: Estimation of cell cycle phases of IE cells based on the labelling
pattern of MC
The labelled precursor was injected on day 6 after lentectomy and the percentage of labelling of the MC as a function of time from the injection was
obtained in four compartments of IE (Fig. 2A-D). In all compartments nulabelled MC were found up to 8 h. The labelled MC began to appear between
4 and 8 h, and completely replaced unlabelled MC from 12 to 24 h, after which
the unlabelled MC started to reappear so that the percentage of labelled MC
gradually decreased by 40-48 h. Although a subsequent increase of the percentage of labelled MC showed slight differences among the various compartments, around 64 h the second peak of the labelling percentage was definitely
indicated in all compartments. At later periods a wide scattering of the value
was observed in all compartments. The estimates of cell cycle phases were
computed according to the mathematical procedures outlined in the method
section, and the results are summarized in Table 1. Considerable differences are
found when the means of S and Gl or the estimated cell cycle times are compared
between LFA and non-LFA. However, the difference between LFA-S and nonLFA-S is significant at the 90 % confidence limits but not at the 95 % confidence
limits. Furthermore, the difference between LFA-G1 and non-LFA-Gl is not
significant at both confidence limits. On the other hand, the total cell cycle time
indicates a significant difference between LFA and non-LFA even at the 95 %
confidence limits.
Experiment II: Delayed incorporation of radioactivity
The purpose of this experiment is to check the possibility that in animals
injected peritoneally with labelled precursor, incorporation of radioactivity
continues beyond the primary labelling period which was estimated to be less
than 4 h in this system (Yamada & Roesel, 1968). The host animal was first
injected with [3H]thymidine as in Expt. I, and 6 h later a piece of regenerating
dorsal iris of an uninjected animal was grafted into the host optic cavity (for
details see the method section). The hosts were sacrificed 24 and 120 h after
grafting, and the sections through the eyes were processed for autography
under the condition used in Expt. I. In 12 cases the experiment was successful.
The areas of the grafted dorsal IE, where cell replication should have been
occurring during the experiment, were selected for the following grain-count
analysis. In the first study the grain counts per MC were made as described
earlier, and the resulting histograms were compared with that of the control in
32
EM B 34
32-95
(1-71)
29-37
d-50)
2709
(319)
40-50
(7-41)
Ventral region
Dorsal and ventral regions
Lens-forming area
Non-lens-forming area
21-94
(7-97)
8-50
(3-91)
8-11
(0-54)
7-60
(4-39)
7-94
(0-595)
9-46
0-63)
8-35
(0-586)
7-60
(6-13)
Means
8-69
(1-87)
11-70
(4-50)
Standard
deviation
G2
203
(0-83)
1-02
(11-17)
1-94
(0-835)
213
(101)
1-10
(16-85)
Standard
deviation
29-94
(11-41)
17-69
(22-10)
0-2345 xlO- 6
(01991 x 109)
15-76
(4-24)
1302
(3-08)
11-14
(6-58)
24-84
(5-34)
00010
(5-6 x 104)
Standard
deviation
18-35
(6-51)
9-40
(8-87)
Means
A
Gl
78-55
45-85
50-33
59-65
44-70
Total cell cycle time§
* Figures are given in h.
f Values in parentheses are standard errors of parameter estimates. The large values of the standard errors in relation to the parameter
estimates for some of the parameters (especially the standard deviations) is caused by the large amount of basic variation present in the observed
data and also by the inability of the estimation routine to obtain precise parameter estimates under these conditions for such a complex function
as p{t).
J M i s divided into Gl and G2.
§ Sums of estimated means of all phases of each compartment.
27-70
(3-54)
Means
Dorsal region
Compartments
S
Cell cycle phase %
Table 1. Estimates of cell cycle parameters of various compartments of iris epithelium*^
X
o
m
>
W
w
w
o
3
Dedifferentiating
iris epithelial cells
505
which grains per neural retinal cell of the host under the same autographic
condition were counted. The results are shown in Fig. 3. In the second study,
the grain counts per nuclear slice of both interphase and mitotic cells were done,
and the labelling frequency was computed on the assumption that a slice with
more than 5 grains inclusive was labelled. In the 24 h series, 0-2-0-5 % of nuclear
slices of IE cells were labelled and the maximum counts per slice were 9. In the
120 h series, 4-8-8-3 % of nuclear slices were labelled with the maximum counts
24 h
0
3
0
3
91
6 9
120 h
12
61
31
151
6
9
12 15 18 21
Control
0
3
6
9
12 15 18 21
Numbers of silver grains/MC
Fig. 3. Histograms showing the grain count distribution in the MC in two experimental series of Expt. II and the control series. The lens-regenerating dorsal iris of
non-injected animals was grafted into the optic cavity of host animals injected 6 h
beforehand. The graft was kept in the host optic cavity for 24 or 120 h, and then the
host eye with the graft was processed for autoradiography. The non-dividing neural
retinal cells of the host eye 120 h after injection served as a control.
per slice being 15. In each series ca. 2000 slices were used for collecting the data.
The optimum grain counts of the host corneal epithelial cells presumably
labelled during the primary labelling period were of the order of a 100. Both
studies indicate that beyond 6 h after injection, the optic cavity retains the cell
labelling activity at a low level. Between 24 and 120 h there occurs an increase
in the cellular level of radioactivity as well as an increase in the labelling frequency, suggesting that the cell labelling activity of the optic cavity persists
even beyond 30 h after injection.
32-2
506
T. YAMADA, M. E. ROESEL AND J. J. BEAUCHAMP
DISCUSSION
Temporal relation between cell cycle and dedifferentiation
Assuming that the average cell cycle time is 45 h for the lens-forming cell
population of IE, we can estimate the number of cell cycles passed during and
after the dedifferentiation period, before the cells enter the terminal phase of
lens fiber cells. Those IE cells forming the primary lens fiber cells of the regenerated lens (prospective primary fibers) go through the dedifferentiation phase
from day 5 to day 10. According to the present data this phase starts with the
later part of the 1st cell cycle and is terminated at the end of the 4th cell cycle.
After completion of dedifferentiation, the prospective primary fibers proceed
one or two more cell cycles before entering the terminal phase of fiber differentiation where accumulation of lens crystallins occurs without replication
of DNA. Concerning the formation of secondary lens fiber cells, some assumptions are needed for making a similar estimation. If we assume that all prospective secondary fibers enter the cell cycle simultaneously with the prospective
primary fibers, their dedifferentiation is extended over eight cell cycles. But
it is possible that the prospective secondary fibers enter the cell cycle later than
day 4. If this is the case, they should have a smaller number of cell cycles for
dedifferentiation.
Comparison of cell cycle parameters of IE cells and those
of depigmented cells derived from them
The estimates of cell cycle parameters obtained here can be compared with
earlier data on the depigmented cell population which is derived from the LFA.
Eisenberg-Zalik & Yamada (1967) reported the estimates for the average
durations of S and G2 of depigmented cells in the lens vesicle 15 days after
lentectomy of adult Notophthalmus viridescens as 19 h and 2 h respectively.
Those experiments were conducted under conditions closely comparable to the
present ones, including the ambient temperature. Mitashov (1969) studied
cell-cycle parameters of lens epithelial cells of lens regenerates which were
formed 14-16 days after removal of retina and lens from adult Triturus cristatus.
The mean durations of S, G2, and the total cell cycle time were estimated as
16, 2 and 23-5 h respectively. These studies applied the graphical method on
the curve of percentage labelled mitoses. From the comparison of these two
sets of figures with the corresponding figures obtained for LFA in the present
work, it is clear that the average values for cell cycle parameters of both depigmented cell populations are considerably shorter than the corresponding values
for pigmented cells of LFA reported here. Since statistics are not available for
the two cited studies, no statistical evaluation of the comparison is possible.
However, from the practical point of view the above comparison suggests the
possibility that dedifferentiation of IE cells is followed by shortening of cell
cycle phases. It is worthwhile to study this point more carefully with a specially
designed experiment.
Dedifferentiating
iris epithelial cells
507
The cell cycle parameters of newt iris epithelial cells cultured in vitro were
measured by Horstman & Zalik (1974). The average duration of Gl, S, G2 and
M were estimated to be 25, 36, 6 and 1-8 h respectively with a total cell cycle
time of 69 h. These estimates are very close to the corresponding values of nonLFA in the present work. It should be pointed out that in both studies the same
species is used, but the ambient temperature for the cell culture was 24 °C,
2-3° higher than that used in the present experiment.
Coupling of cell proliferation and dedifferentiation
As clear from the first paragraph of the discussion, dedifTerentiation of IE
cells is completed while the cells are in the cell cycle induced by lentectomy.
Studies of experimental intervention of lens regeneration support the notion that
dedifferentiation is dependent upon the proliferation of IE cells. Lens regeneration is known to be sensitive to X-radiation (Politzer, 1930), and the target of
the radiation in this system is the iris (Donaldson, 1972). It has been recently
demonstrated that X-radiation inhibits proliferation of IE cells in situ after
lentectomy or when cultured in vitro (Michel & Yamada, 1974). Complete
inhibition of lens regeneration by repeated injection of actinomycin D (Yamada
& Roesel, 1964) is probably due to suppression of re-entry of IE cells into the
cell cycle. Since both X-radiation and actinomycin suppress depigmentation of
IE cells along with cell proliferation, it is probable that inhibition of dedifferentiation caused by inhibition of cell proliferation is the reason for suppressed
lens regeneration.
How cell proliferation is coupled with dedifferentiation should be the next
issue to be raised. In this connexion one open question is whether simple dilution
of melanosomes caused by cell replication is sufficient to account for the observed depigmentation of IE cells. Assuming that no production nor degradation of melanosomes in IE cells occurs during the depigmentation period (see
Introduction) and using a preliminary estimate of the melanosome number per
normal IE cells (6000), the minimum of four cell cycles undergone by LFA
cells before complete depigmentation is judged insufficient to cause complete
depigmentation by simple dilution. This is in conformity with the notion that
melanosomes are discharged from IE cells in the cell cycle as discussed in the
Introduction.
As discussed, IE cells of the LFA go through complete depigmentation and
lens differentiation, while those of non-LFA do not complete depigmentation
and revert to the normal condition of IE cells. In the present data, the total cell
cycle time of LFA cells is significantly smaller than that of non-LFA cells.
Therefore during the time the mean LFA cells complete four cell cycles, the
minimum number of cell cycles needed for depigmentation, the mean non-LFA
cells progress only 2-4 cell cycles. Even if we assume that melanosomes are discharged evenly in LFA and non-LFA cells, there is the possibility that the
difference in dilution of melanosomes by cell replication decides the alternative
508
T. YAMADA, M. E. ROESEL AND J. J. BEAUCHAMP
of complete or incomplete depigmentation at a critical time. Since melanosome
discharge has been observed only when IE cells are in the cell cycle, it is possible that the discharge like many other cellular functions is related to the cell
cycle time in such a way that the shorter the cell cycle time the more is accomplished per unit time. Thus it seems probable that in this system the alternative
pathways of dedifferentiation of IE cells and hence cell-type conversion is
controlled by the differential cell cycle time.
Persistence of cell labelling activity in the optic cavity
The use of the percentage of labelled mitoses for estimation of cell cycle
parameters in vivo presupposes that cell labelling is limited to a time interval
which is relatively short compared with S and immediately follows injection.
However, there have been reports indicating that these conditions may not be
fully realized (Galassi, 1967; Rafferty & Gfeller, 1970). According to RafTerty
& Gfeller (1970), frog lenses from non-radioactive donors grafted into the
optic cavity of frogs injected with [3H]thymidine 5-72 h beforehand, show
radioactivity detectable by autoradiography. The inquiries of those authors
suggest that in the delayed incorporation, radioactivity is mediated by a factor
of high molecular weight present in serum and aqueous humor. The results of
Expt. II demonstrate that in the present system cell labelling in the optic cavity
also persists for a long time, although at a very low level, after termination of
the primary labelling time which was earlier estimated as less than 4 h (Yamada
& Roesel, 1968).
This research was sponsored by the U.S. Atomic Energy Commission under contract with
Union Carbide Corporation and by Fonds National Suisse de la Recherche Scientifique
(Request no. 3.0860.73). A part of the research was conducted while T. Y. was the recipient
of a senior fellowship from the European Molecular Biology Organization. The authors
gratefully acknowledge the support of those organizations.
Completion of the present work was only possible with the help provided by Dr V. R. R.
Uppuluri, Mr Peter Thall, and Mr Ronald Johnson in the mathematical treatment of the
data, and the authors express deep appreciation of their cooperation. The authors are also
thankful to Mrs Lola M. Kyte for her excellent technical assistance. They further acknowledge critical reading of the manuscript by Dr Nikolai Odarchenko, Dr James N. Dumont
and Dr Sohan P. Modak.
The paper is dedicated to Professor Etienne Wolff on his retirement.
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{Received 9 April 1975, revised 19 May 1975)