Cell cycle parameter ins dedifferentiating iris epithelial cells · Embryol. exp. Morph. Vol. 34,...

/ . Embryol. exp. Morph. Vol. 34, 2, pp. 497-5JO, 1975 4 9 7

Printed in Great Britain

Cell cycle parameters in dedifferentiatingiris epithelial cells

ByTUNEO YAMADA,1 MARION E. ROESEL2 ANDJOHN J. BEAUCHAMP3

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 CarbideCorporation, Nuclear Division, Oak Ridge, Tennessee

SUMMARYAfter 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 thecell cycle, and then differentiate into lens cells. The remaining iris epithelial cells becomepartially depigmented in the induced cell cycle, but resynthesize melanosomes and recoverthe normal state of iris epithelial cells. The two groups of cells are spatially separated withinthe iris epithelium. The cell cycle parameters of both groups of iris epithelial cells were esti-mated by a mathematical procedure on a computerized programme from the percentage oflabelled mitotic cells as a function of time after peritoneal injection of [3H]methyl-thymidineon day 6 after lentectomy. The total cell cycle time was found significantly shorter in the cellpopulation 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 dedif-ferentiation was discussed. Grafting of unlabelled iris into the optic cavity of host animalsinjected 6 h beforehand with [3H]methyl-thymidine, followed by a study of radioactivity ofiris epithelial cells of the graft demonstrated incorporation at a low level during the wholeperiod of the experiment in which the cell cycle parameters were estimated. The data usedfor the estimation were corrected for the delayed incorporation.

INTRODUCTION

Lentectomy of the adult newt eye is followed by regeneration of a functionallens. The classical notion that this lens is derived from the iris epithelium (IE)was supported by a considerable amount of circumstantial evidence (for reviewYamada, 1967). Recent tissue culture experiments (Connelly, Ortiz & Yamada,1973; Yamada, Reese & McDevitt, 1973; Yamada & McDevitt, 1974) haveprovided a direct proof for the notion. This implies that upon lentectomy, fullydifferentiated 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 1011Lausanne, 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 whichthey do not follow during their ontogenesis. The whole sequence of events, whichis induced by lentectomy in the IE cells can be divided into three processes: induc-tion of cell replication, dedifferentiation, and redifferentiation (Yamada, 1972).The earliest cellular change observed after lentecomy in IE cells is activationof 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 afterlentectomy (Yamada & Roesel, 1971). While the IE cells are in the cell cycle,the population of melanosomes is progressively eliminated - a process tradition-ally called depigmentation. After depigmentation has been completed, the cellsform a lens vesicle and withdraw from the cell cycle as they differentiate intolens fiber cells (Eisenberg & Yamada, 1966; Reyer, 1971; Eguchi & Shingai,1971). Since completion of depigmentation in IE cells coincides in time andlocation with the appearance of competence for lens formation, one can arguethat during the depigmentation phase the cells not only lose their overt differen-tiation but also become disengaged from their original commitment. Hence thephase of depigmentation can be identified with the phase of dedifferentiation.

Depigmentation of IE cells appears to be caused mainly by discharge ofmelanosomes from the cell body (Wolff, 1895; Eguchi, 1963). The most impor-tant mode of discharge is separation from the cell of a package of melanosomesbound by membrane (Dumont & Yamada, 1972, unpublished). During thedepigmentation phase which lasts for 6 days or more, the surface of IE cellssends out extensive projections, and the IE becomes infiltrated by macrophagesand neutrophils which take up the discharged materials (Eguchi, 1963; Yamada& Dumont, 1972). On the other hand, the absence of premelanosomes in IEcells undergoing active replication induced by lentectomy implies that synthesisof melanosomes is not coupled with cell replication. Hence during depigmenta-tion, cell replication should also contribute to reduction in the melanosomenumber per cell by dilution.

One more aspect needed as background information in the present paper isrelated to the fact that cell replication is induced all over the iris ring by lentec-tomy, but that only some of those proliferating IE cells become transformedinto the lens cells. The discussion made in the preceding paragraph concernsthis latter population of IE cells. The remaining IE cells induced in replicationgo through a process of partial depigmentation, resynthesize melanosomes, andresume 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: oneinvolves 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 stateof IE cells. So far as lens regeneration in situ is concerned, the choice of pathwaysis determined by the location of cells within the IE. This also applies to repeatedlens regeneration induced by repeated lentectomy.

In the efforts to elucidate the nature of the dedifferentiation process by variousapproaches (Zalik & Scott, 1972, 1973; Ortiz, Yamada & Hsie, 1973; Idoyaga-Vargas & Yamada, 1974), information on the cell cycle parameters of IE cellsduring lens regeneration in situ is indispensable. The main part of this paperconcerns estimation of the duration of cell cycle phases of IE cells involved inthe alternative pathways of dedifferentiation during lens regeneration in situ. Theautoradiographic data on the percentage of labelled mitoses as a function oftime after peritoneal injection of tritiated thymidine into the adult newt 6 daysafter lentectomy were used for the basis of computation. At this time the IEcells begin active depigmentation and the strict synchronization in the DNAsynthesis, which is characteristic for the preceding period but inconvenient forthe 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 timespent in these three phases follows a truncated normal distribution. An expressionhas been derived to describe p(t), which is the percentage labelled cells in themitotic phase at successive times t. Since p{t) is a non-linear function of sixunknown parameters (averages and variances for the distributions of timesspent 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 obtainthe weighted least-square estimates of these quantities.

In another group of experiments grafting of iris tissue and autoradiographywere combined to test the possible persistence of cell labelling activity in thepresent system. A low level of cell labelling activity in the optic environment wasdemonstrated after the end of the primary labelling period due to injectionof labelled precursor. The percentage of labelled mitotic cells (MC) used toestimate 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 springwater in laboratory tanks at 21-22 °C under continuous illumination wereexclusively used.

Lentectomy. The animals were anesthetized with Tricaine (Sigma), a paperplug was inserted into the buccal cavity, and a horizontal incision was made inthe cornea. The lens was removed bilaterally through the incision by a gentlepush on the dorsal and ventral areas of the cornea.

Cell labelling and autoradiography. In experiment I the lentectomized animals


were injected intraperitoneally with 3 /*Ci/g body weight of [3H]methyl-thymidine(specific activity 14 Ci/mmole, Schwarz BioResearch) 6 days after lent-ectomy. The animals were sacrificed at 4 or 8 h intervals during the periodof 4-112 h after injection. Subsequent to fixation of the head in Carnoy, theeyeballs were separated, embedded in paraffin, and sectioned serially at 5 [ivs\thickness. The orientation of the sections was sagittal to the eye as an indepen-dent bilateral body. The mounted sections were treated with 10 % hydrogenperoxide for 22 h to achieve partial bleaching of melanin. The possible effectsof the bleaching procedure on autoradiographic counts was checked and foundinsignificant. The sections were covered with NTB 3 emulsion (Kodak), exposedfor 2 weeks at 4 °C, and developed with D 11 developer (Kodak) for 3 min at20 °C. Mayer's hemalum was used to stain chromatin.

DR VR LFA Non-LFA

d e

Fig. 1. Compartments of iris epithelium used in Expt. I. (A, B) diagrams of theeye 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 stromaare not taken into account. (C) Dorsal iris epithelium composed of LFA anddorsal non-LFA. d-d and e-e indicate the levels of the saggital section of IEshown in (D) and (E) respectively. In (D) the dorsal one-half of the inner lamina andthe distal one-fourth of the outer lamina of IE are indicated as LFA. In (E) thedorsal one third of the inner lamina alone belongs to LFA. Diagrams (B-D) are basedon autoradiographic tracing of lens-forming cells (Eisenberg & Yamada, 1966;Reyer, 1971; Eguchi & Shingai, 1971), and are comparable to the embryological fatemaps. But in the absence of data from localized vital staining and genetic mosaicsthe 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 asdorsal region (DR), ventral region (VR), lens-forming area (LFA) and non-lens-forming area (non-LFA) as explained in Fig. 1. LFA is a part of DR,


while non-LFA comprises the remaining part of DR and the whole VR. Thecells which participate in lens cell differentiation after complete depigmentationare all located in LFA, while the cells of non-LFA retain IE specificity afterincomplete depigmentation.

Methods for counting labelled and unlabelled MC. In Expt. I, the completeset of serial tissue sections was scanned under a microscope, and in eachcompartment of IE, the total numbers of labelled and unlabelled MC wererecorded. One mitotic IE cell is sectioned into 2-4 consecutive slices. Since inthe sectioned IE the nuclei are widely dispersed, and the number of IE cellsper tissue section is limited, it was possible to follow a single mitotic cell throughthe serial tissue sections. The grain counts of single MC were obtained by addi-tion of silver grains over all slices of the cell. Since Expt. II demonstratedprogressive low-level labelling of cells in cell cycle during the time intervalemployed in Expt. I, it became necessary to consider the delayed labelling inour method of distinguishing labelled and unlabelled MC. This was done byusing the following set of minimum grain counts per MC in determination oflabelled MC: 8 grains for 4-20 h series; 10 grains for 24-44 h series. 11 grainsfor 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 theoptic cavity, which demonstrated that the incorporation is a function of thetime interval during which the cells are exposed to the environment.

Estimation of cell cycle parameters. For convenience, we use the followingnotation 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 veryshort, 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 beenshown (Okumura, Onozawa, Morita & Matsuzawa, 1973) that the expressionfor p(t) involves the convulution of the distributions for Xx, X2, X3. In ourmodelling effort truncated normal distributions, which do not allow negativevalues of our random variables as normal distributions, would have been usedfor Xlt X2 and X3.

The observed data, p(t), are given as the proportion of labelled cells in M atsuccessive times /, that is

where 1 -> (t) is the observed number of labelled MC at time / and N(t) isthe observed number of MC at time t. The expression for p(t) involves sixunknown parameters (three unknown mean or average times spent in the phasesand three unknown variances) of the distributions for Xlt X2 and X3.

The weighted-least-squares estimates of these parameters were obtained byminimizing ^

alH


where w(t) is the weight associated with the observation p(t) at time /. In thisstudy w(t) was chosen equal to N(t)![p(t)(\—/p(t))] under the assumption thatthe observed values p(t) followed a binomial distribution. Since p(t) is a non-linear function of the six unknown parameters, an iterative estimation techniquewas used to obtain the estimates in Table 1. The computer program used wasthe program N0NLS2 written by Wesley & Watts (1970) based on the non-linearestimation technique of Marquardt (1963).

0 20t 40 t 60 • 80 h00112 h7d 8d 9d 10 d

8

cell

s

o

toti

E

—L

abel

100 -

80-

60-

40-

20 -

f«MMI

T i l U '

MM •

•

•

•

•

:

B. VR

•• •

, * . •

* ' * t•

0 20* 40 • 60 • 80 h00H2 h7d 8d 9d 10 d

88uO

1-au

bell

3

100 -

80-

60-

40-

20-

— . C. LFA

i . •• • • •, • • • •

• • • : •• • . • • •

• • • • • •

• ••• •

i '20' 40 ' 60 f 80 '100112

7d 8d 9d 10 d

100-

80--

60--

40-

20-

I M M H I *

• •

• #

•

f- i i . 1 : i l i

D. Non-LFA

•

• • •r•

••• • " :• ••

*

20' 40 ' 60 ' 80 M00112 h7d 8d 9 d lOd

Fig. 2. The percentage of labelled mitotic cells of the four compartments of iris epi-thelium as a function of time after injection. On the abscissa, h signifies hoursafter 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 wasinjected peritoneally with 3 /*Ci/g body weight with [3H]methyl-thymidine, andlentectomized through a U-shaped incision in the cornea. The shape of theincision was selected to facilitate retention of the graft subsequently implantedinto the optic cavity. Six h after injection the regenerating lenses were removedfrom 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


fixed in the Carnoy fixative, and the eyes were embedded in paraffin andsectioned serially into sections 5 /im thick. The mounted sections were processedfor autoradiography as described above.

RESULTS

Experiment I: Estimation of cell cycle phases of IE cells based on the labellingpattern of MC

The labelled precursor was injected on day 6 after lentectomy and the per-centage of labelling of the MC as a function of time from the injection wasobtained in four compartments of IE (Fig. 2A-D). In all compartments nu-labelled MC were found up to 8 h. The labelled MC began to appear between4 and 8 h, and completely replaced unlabelled MC from 12 to 24 h, after whichthe unlabelled MC started to reappear so that the percentage of labelled MCgradually decreased by 40-48 h. Although a subsequent increase of the per-centage of labelled MC showed slight differences among the various compart-ments, around 64 h the second peak of the labelling percentage was definitelyindicated in all compartments. At later periods a wide scattering of the valuewas observed in all compartments. The estimates of cell cycle phases werecomputed according to the mathematical procedures outlined in the methodsection, and the results are summarized in Table 1. Considerable differences arefound when the means of S and Gl or the estimated cell cycle times are comparedbetween LFA and non-LFA. However, the difference between LFA-S and non-LFA-S is significant at the 90 % confidence limits but not at the 95 % confidencelimits. Furthermore, the difference between LFA-G1 and non-LFA-Gl is notsignificant at both confidence limits. On the other hand, the total cell cycle timeindicates 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 animalsinjected peritoneally with labelled precursor, incorporation of radioactivitycontinues beyond the primary labelling period which was estimated to be lessthan 4 h in this system (Yamada & Roesel, 1968). The host animal was firstinjected with [3H]thymidine as in Expt. I, and 6 h later a piece of regeneratingdorsal iris of an uninjected animal was grafted into the host optic cavity (fordetails see the method section). The hosts were sacrificed 24 and 120 h aftergrafting, and the sections through the eyes were processed for autographyunder 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 beenoccurring during the experiment, were selected for the following grain-countanalysis. In the first study the grain counts per MC were made as describedearlier, and the resulting histograms were compared with that of the control in

32 EM B 34

Tab

le

1. E

stim

ates

of

cel

l cy

cle

para

met

ers

of v

ario

us c

ompa

rtm

ents

of

iri

s ep

ithe

lium

*^

Co

mp

artm

ents

Do

rsal

reg

ion

Ven

tral

reg

ion

Do

rsal

an

d ve

ntra

l re

gio

ns

Len

s-fo

rmin

g ar

ea

Non

-len

s-fo

rmin

g ar

ea

Mea

ns

27-7

0(3

-54)

32-9

5(1

-71)

29-3

7d

-50

)

27

09

(31

9)

40-5

0(7

-41)

S

Sta

ndar

dde

viat

ion

11-7

0(4

-50)

8-69

(1-8

7)

9-46

0-63

)

8-50

(3-9

1)

21-9

4(7

-97)

Cel

l cy

cle

phas

e %

G2

Mea

ns

7-60

(6-1

3)

8-35

(0-5

86)

7-94

(0-5

95)

7-60

(4-3

9)

8-11

(0-5

4)

Sta

ndar

dde

viat

ion

1-10

(16-

85)

21

3(1

01)

1-94

(0-8

35)

1-02

(11-

17)

20

3(0

-83)

Mea

ns

9-40

(8-8

7)

18-3

5(6

-51)

13

02

(3-0

8)

11-1

4(6

-58)

29-9

4(1

1-41

)

Gl A

Sta

ndar

dde

viat

ion

0001

0(5

-6 x

104 )

24-8

4(5

-34)

15-7

6(4

-24)

0-23

45 x

lO-6

(019

91 x

109 )

17-6

9(2

2-10

)

Tot

al c

ell

cycl

e ti

me§

3 o w w

44-7

0

59-6

5

50-3

3

45-8

5

78-5

5

* F

igu

res

are

give

n in

h.

f V

alue

s in

par

enth

eses

are

sta

nd

ard

erro

rs o

f p

aram

eter

est

imat

es.

The

lar

ge v

alue

s of

the

sta

ndar

d er

rors

in

rela

tion

to

the

para

met

eres

tim

ates

for

som

e of

th

e p

aram

eter

s (e

spec

iall

y th

e st

and

ard

devi

atio

ns)

is c

ause

d by

the

larg

e am

ount

of

basi

c va

riat

ion

pres

ent

in t

he o

bser

ved

dat

a an

d al

so b

y th

e in

abil

ity

of t

he

esti

mat

ion

rou

tin

e to

obt

ain

prec

ise

para

met

er e

stim

ates

und

er t

hese

con

diti

ons

for

such

a c

ompl

ex f

unct

ion

as p

{t).

J M

is d

ivid

ed i

nto

Gl

and

G2

.§

Sum

s of

est

imat

ed m

ean

s of

all

pha

ses

of e

ach

com

par

tmen

t.

W m > o X


which grains per neural retinal cell of the host under the same autographiccondition 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 withmore than 5 grains inclusive was labelled. In the 24 h series, 0-2-0-5 % of nuclearslices of IE cells were labelled and the maximum counts per slice were 9. In the120 h series, 4-8-8-3 % of nuclear slices were labelled with the maximum counts

9 1

6 1

3 1

151

24 h

0 3 6 9 12120 h

0 3 6 9 12 15 18 21

Control

0 3 6 9 12 15 18 21Numbers of silver grains/MC

Fig. 3. Histograms showing the grain count distribution in the MC in two experi-mental series of Expt. II and the control series. The lens-regenerating dorsal iris ofnon-injected animals was grafted into the optic cavity of host animals injected 6 hbeforehand. The graft was kept in the host optic cavity for 24 or 120 h, and then thehost eye with the graft was processed for autoradiography. The non-dividing neuralretinal 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 presumablylabelled during the primary labelling period were of the order of a 100. Bothstudies indicate that beyond 6 h after injection, the optic cavity retains the celllabelling activity at a low level. Between 24 and 120 h there occurs an increasein the cellular level of radioactivity as well as an increase in the labelling fre-quency, suggesting that the cell labelling activity of the optic cavity persistseven beyond 30 h after injection.

32-2


DISCUSSION

Temporal relation between cell cycle and dedifferentiation

Assuming that the average cell cycle time is 45 h for the lens-forming cellpopulation of IE, we can estimate the number of cell cycles passed during andafter the dedifferentiation period, before the cells enter the terminal phase oflens fiber cells. Those IE cells forming the primary lens fiber cells of the regen-erated lens (prospective primary fibers) go through the dedifferentiation phasefrom day 5 to day 10. According to the present data this phase starts with thelater 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 proceedone or two more cell cycles before entering the terminal phase of fiber dif-ferentiation where accumulation of lens crystallins occurs without replicationof DNA. Concerning the formation of secondary lens fiber cells, some assump-tions are needed for making a similar estimation. If we assume that all prospec-tive secondary fibers enter the cell cycle simultaneously with the prospectiveprimary fibers, their dedifferentiation is extended over eight cell cycles. Butit is possible that the prospective secondary fibers enter the cell cycle later thanday 4. If this is the case, they should have a smaller number of cell cycles fordedifferentiation.

Comparison of cell cycle parameters of IE cells and thoseof depigmented cells derived from them

The estimates of cell cycle parameters obtained here can be compared withearlier data on the depigmented cell population which is derived from the LFA.Eisenberg-Zalik & Yamada (1967) reported the estimates for the averagedurations of S and G2 of depigmented cells in the lens vesicle 15 days afterlentectomy of adult Notophthalmus viridescens as 19 h and 2 h respectively.Those experiments were conducted under conditions closely comparable to thepresent ones, including the ambient temperature. Mitashov (1969) studiedcell-cycle parameters of lens epithelial cells of lens regenerates which wereformed 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 as16, 2 and 23-5 h respectively. These studies applied the graphical method onthe curve of percentage labelled mitoses. From the comparison of these twosets of figures with the corresponding figures obtained for LFA in the presentwork, it is clear that the average values for cell cycle parameters of both depig-mented cell populations are considerably shorter than the corresponding valuesfor pigmented cells of LFA reported here. Since statistics are not available forthe two cited studies, no statistical evaluation of the comparison is possible.However, from the practical point of view the above comparison suggests thepossibility that dedifferentiation of IE cells is followed by shortening of cellcycle phases. It is worthwhile to study this point more carefully with a speciallydesigned experiment.


The cell cycle parameters of newt iris epithelial cells cultured in vitro weremeasured by Horstman & Zalik (1974). The average duration of Gl, S, G2 andM were estimated to be 25, 36, 6 and 1-8 h respectively with a total cell cycletime of 69 h. These estimates are very close to the corresponding values of non-LFA in the present work. It should be pointed out that in both studies the samespecies 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 IEcells is completed while the cells are in the cell cycle induced by lentectomy.Studies of experimental intervention of lens regeneration support the notion thatdedifferentiation is dependent upon the proliferation of IE cells. Lens regenera-tion is known to be sensitive to X-radiation (Politzer, 1930), and the target ofthe radiation in this system is the iris (Donaldson, 1972). It has been recentlydemonstrated that X-radiation inhibits proliferation of IE cells in situ afterlentectomy or when cultured in vitro (Michel & Yamada, 1974). Completeinhibition of lens regeneration by repeated injection of actinomycin D (Yamada& Roesel, 1964) is probably due to suppression of re-entry of IE cells into thecell cycle. Since both X-radiation and actinomycin suppress depigmentation ofIE cells along with cell proliferation, it is probable that inhibition of dedif-ferentiation caused by inhibition of cell proliferation is the reason for suppressedlens regeneration.

How cell proliferation is coupled with dedifferentiation should be the nextissue to be raised. In this connexion one open question is whether simple dilutionof melanosomes caused by cell replication is sufficient to account for the ob-served depigmentation of IE cells. Assuming that no production nor degrada-tion of melanosomes in IE cells occurs during the depigmentation period (seeIntroduction) and using a preliminary estimate of the melanosome number pernormal IE cells (6000), the minimum of four cell cycles undergone by LFAcells before complete depigmentation is judged insufficient to cause completedepigmentation by simple dilution. This is in conformity with the notion thatmelanosomes are discharged from IE cells in the cell cycle as discussed in theIntroduction.

As discussed, IE cells of the LFA go through complete depigmentation andlens differentiation, while those of non-LFA do not complete depigmentationand revert to the normal condition of IE cells. In the present data, the total cellcycle 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, theminimum number of cell cycles needed for depigmentation, the mean non-LFAcells progress only 2-4 cell cycles. Even if we assume that melanosomes are dis-charged evenly in LFA and non-LFA cells, there is the possibility that thedifference in dilution of melanosomes by cell replication decides the alternative


of complete or incomplete depigmentation at a critical time. Since melanosomedischarge has been observed only when IE cells are in the cell cycle, it is pos-sible that the discharge like many other cellular functions is related to the cellcycle time in such a way that the shorter the cell cycle time the more is accom-plished per unit time. Thus it seems probable that in this system the alternativepathways of dedifferentiation of IE cells and hence cell-type conversion iscontrolled 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 cycleparameters in vivo presupposes that cell labelling is limited to a time intervalwhich is relatively short compared with S and immediately follows injection.However, there have been reports indicating that these conditions may not befully realized (Galassi, 1967; Rafferty & Gfeller, 1970). According to RafTerty& Gfeller (1970), frog lenses from non-radioactive donors grafted into theoptic cavity of frogs injected with [3H]thymidine 5-72 h beforehand, showradioactivity detectable by autoradiography. The inquiries of those authorssuggest that in the delayed incorporation, radioactivity is mediated by a factorof high molecular weight present in serum and aqueous humor. The results ofExpt. II demonstrate that in the present system cell labelling in the optic cavityalso persists for a long time, although at a very low level, after termination ofthe 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 withUnion 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 recipientof a senior fellowship from the European Molecular Biology Organization. The authorsgratefully 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 thedata, and the authors express deep appreciation of their cooperation. The authors are alsothankful to Mrs Lola M. Kyte for her excellent technical assistance. They further acknow-ledge critical reading of the manuscript by Dr Nikolai Odarchenko, Dr James N. Dumontand Dr Sohan P. Modak.

The paper is dedicated to Professor Etienne Wolff on his retirement.

REFERENCES

CONNELLY, T. C, ORTIZ, J. R. & YAMADA, T. (1973). Influence of the pituitary on Wolffianlens regeneration. Devi Biol. 13, 301-315.

DONALDSON, D. J. (1972). Effects of X-irradiation on lens regeneration in adult Triturusviridescens. Anat. Rec. 172, 45-56.

DUMONT, J. N., YAMADA, T. & CONE, M. V. (1970). Alteration of nucleolar ultrastructure iniris epithelial cells during initiation of Wolffian lens regeneration. /. exp. Zool. 174, 187—204.

DUMONT, J. N. & YAMADA, T. (1972). Dedifferentiation of iris epithelial cells. Devi Biol.29, 385-401.

Dedifferentiating iris epithelial cells 509EGUCHI, G. (1963). Electron microscopic studies on lens regeneration. I. Mechanism of

depigmentation of the iris. Embryologia 8, 47-62.EGUCHI, G. & SHINGAI, R. (1971). Cellular analysis on localization of lens-forming potency

in the newt iris epithelium. Development, Growth, Diff. {Japan) 13, 337-349.EISENBERG, S. & YAMADA, T. (1966). A study of DNA synthesis during transformation of the

iris into lens in the lentectomized newt. / . exp. Zool. 162, 353-367.EISENBERG-ZALIK, S. & YAMADA, T. (1967). The cell cycle during lens regeneration. / . exp.

Zool. 165, 385-394.GALASSI, L. (1967). Delayed and direct labelling after a systemic injection of thymidine-3H.

/ . Histoch. Cytochem. 15, 565-579.HORSTMAN, L. P. & ZALIK, S. E. (1974). Growth of newt iris epithelial cells in vitro: A study

of cell cycle. Expl Cell Res. 84, 1-14.IDOYAGA-VARGAS, V. & YAMADA, T. (1974). Glucosaminidase and dedifferentiation of newt

iris epithelium. Differentiation 2, 91-98.JAUKER, F. & YAMADA, T. (1973). Progressive alteration in the pattern of nucleic acid

metabolism in the newt iris in cultivation. / . exp. Zool. 183, 145-152.KARASAKI, S. (1964). An electron microscopic study of Wolffian lens regeneration in the adult

newt. / . Ultrastruct. Res. 11, 246-273.MARQUARDT, D. W. (1963). An algorithm for least-squares estimation of nonlinear para-

meters. J. Siam 11, 431-441.MICHEL, C. & YAMADA, T. (1974). Cellular studies of X-ray induced inhibition of lens

regeneration. Differentiation 2, 193-201.MITASHOV, V. I. (1969). Characteristics of the mitotic cycle during lens regeneration in an

experiment involving complete removal of the retina and lens in adult newts (Trituruscristatus). Dokl. (Proc.) Acad. Sci. U.S.S.R. {Biol. Sci.) 189, 834-836.

OKUMURA, Y., ONOZAWA, M., MORITA, T. & Matsuzawa, T. (1973). A simple method forestimation of cell cycle parameters. Expl Cell Res. 78, 233-236.

ORTIZ, J. R., YAMADA, T. & HSIE, A. W. (1973). Induction of the stellate configuration incultured iris epithelial cells by adenosine and compounds related to adenosine 3':5'-cyclicmonophosphate. Proc. natn. Acad. Sci, U.S.A. 70, 2286-2290.

POLITZER, G. (1930). Uber den Einfluss der Rontgenstrahlen auf die Regeneration der Linse.Z. wiss. Biol. {Abt. Roux Arch. EntwMech. Org.) 121, 39-71.

RAFFERTY, N. S. & GFELLER, E. (1970). 3H-thymidine mediated and delayed nuclear labelingin frog lens epithelium. Expl Cell Res. 59, 249-258.

REESE, D. H. (1973). In vitro initiation in the newt iris of some early molecular events of lensregeneration. Expl Eye Res. 17, 435-444.

REESE, D. H., PUCCIA, E. & YAMADA, T. (1969). Activation of ribosomal RNA synthesis ininitiation of Wolffian lens regeneration. / . exp. Zool. 170, 259-268.

REYER, R. W. (1971). DNA synthesis and incorporation of labeled iris cells into the lensduring lens regeneration in adult newts. Devi Biol. 24, 533-558.

WESLEY, G. W. & WATTS, J. A. (1970). The computing technology center numerical analysislibrary. Report CTC-39 Union Carbide Nuclear Division, Oak Ridge, Tennessee, October1970, pp. 356-382.

WOLFF, F. (1895). Entwickelungsphysiologische Studien. Arch. EntwMech. Org. 1, 380-390.YAMADA, T. (1967). Cellular and subcellular events in Wolffian lens regeneration. Cur. Top.

Devi Biol. 2, 247-283.YAMADA, T. (1972). Control mechanisms in cellular metaplasia. Proc. 1st Int. Conf. Cell

Differentiation, pp. 56-60.YAMADA, T. & DUMONT, J. N. (1972). Macrophage activity in Wolffian lens regeneration.

/ . Morph. 136, 367-384.YAMADA, T. & MCDEVITT, D. S. (1974). Direct evidence for transformation of differentiated

iris epithelial cells into lens cells. Devi Biol. 38, 104-118.YAMADA, T., REESE, D. H. & MCDEVITT, D. S. (1973). Transformation of iris into lens in

vitro and its dependency on neural retina. Differentiation 1, 65-82.YAMADA, T. & ROESEL, M. E. (1964). Effects of actinomycin D on the lens regenerating

system. / . Embryol. exp. Morph. 12, 713-725.


YAMADA, T. & ROESEL, M. E. (1968). Labeling of lens regenerate cells grafted into the newtoptic chamber. A study of availability time of tritiated thymidine. Expl Cell Res. 50,649-652.

YAMADA, T. & ROESEL, M. E. (1969). Activation of DNA replication in the iris epitheliumby lens removal. / . exp. Zool. 171, 425-431.

YAMADA, T. & ROESEL, M. E. (1971). Control of mitotic activity in Wolffian lens regeneration.J. exp. Zool. 117, 119-128.

ZALIK, S. E. & SCOTT, V. (1972). Cell surface change during dedifferentiation in the meta-plastic transformation of iris into lens. / . Cell Biol. 55, 134-146.

ZALIK, S. E. & SCOTT, V. (1973). Sequential disappearance of cell surface components duringdedifferentiation in lens regeneration. Nature New Biology 244, 212-214.

{Received 9 April 1975, revised 19 May 1975)

Cell cycle parameter ins dedifferentiating iris epithelial cells · Embryol. exp. Morph. Vol. 34,...

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