/. Embryo!, e.xp. Morpli. Vol. 30, 1, pp. 63-71, 1973
63
Printed in Great Britain
Elicitation of lens formation
from the 'ventral iris' epithelium of the newt by a
carcinogen, iV-methyl-iV'-nitro-iV-nitrosoguanidine
By GORO EGUCHP AND KENJI WATANABE 1
From the Laboratory for Differentiation and Morphogenesis,
Institute of Biophysics, University of Kyoto
SUMMARY
A potent carcinogenic compound, iV-methyl-./V'-nitro-./V-nitrosoguanidine (NG), was
administered to the lentectomized eyes of the newt, Triturus pyrrhogaster. Supernumerary
lenses were formed from the dorsal iris and even from the ventral iris, which never transforms
into lens following simple surgical lentectomy or the grafting of isolated iris pieces into lentectomized eyes. After the administration of NG the pigmented epithelial cells of the
ventral iris were induced to gain the competence for transformation into lens cells. The
induced competence of the iris epithelial cells was stably maintained for at least 12 months.
The secondary regeneration of the lens 45 days or 12 months after the primary lentectomy
took place at the same positions of the ventral area of the iris where the primary lens regeneration had been induced by NG.
The effects of NG were also confirmed by implanting isolated pieces of iris treated with NG
solution into lentectomized eyes. Five implants out of 42 available experiments on the ventral
half of the iris were found to be transformed into lens.
INTRODUCTION
The iris epithelium of newts is capable of transforming into lens. When a lens
is surgically removed from the eye, the lens regeneration occurs always only
from the dorsal marginal portion of the iris epithelium. Among a large number
of papers so far published (for reviews see Reyer, 1954, 1962; Scheib, 1965;
Yamada, 1967) there are none indicating the possibility of the transformation
of ventral iris epithelium into lens. When isolated iris pieces are implanted in
lentectomized host eyes, the pieces derived from the ventral half of the iris are
incapable of transformation into a lens, but ones from the dorsal half are capable
of transformation (Sato, 1930, 1951; Mikami, 1941). At the cellular level,
Eguchi & Shingai (1971) have recently suggested that there are two different cell
populations, one being capable and the other incapable of transforming into
lens, and cells of the former type are densely localized in the dorsal part of the
iris. The question may then be asked if there may be any means of eliciting lens
1
Authors' address: Laboratory for Differentiation and Morphogenesis, Institute of Biophysics, Faculty of Science, University of Kyoto, Kyoto 606, Japan.
64
G. EGUCHI AND K. WATANABE
formation from the ventral iris, which mostly consists of cells apparently incapable of transformation.
This paper deals with the finding that the administration of iV-methyl-iV'nitro-JV-nitrosoguanidine (NG) to lentectomized newt eyes successfully elicits
supernumerary lenses from the ventral or the lateral portions of the iris epithelium
in addition to the usual dorsal portion. This drug, NG, is well known as a mutagen.
Its very potent carcinogenic effect has also been shown (Sugimura, Fujimura &
Baba, 1970).
MATERIALS AND METHODS
Adult Japanese newts, Triturus pyrrhogaster, were used as materials for the
present experiment. A small particle of NG was inserted into the lentectomized
eyes. In another series of experiments pieces of iris were treated in the NG
solution and were then implanted into the lentectomized eyes. The procedure
for lentectomy was the same as given in Eguchi & Shingai (1971). The lens
removal was performed on both eyes.
Administration ofNG crystal. A single particle of NG (crystallized NG, K &
K Laboratories, Inc., Plainview, N.Y.) from 15 to 20 fig in weight was placed
into the posterior eye chamber either immediately after or 5 days after the
lentectomy. The inserted NG particle remained at least for 1 h within the eye
chamber.
Implantation of iris pieces treated with NG. The iris ring was obtained by
cutting the irido-corneal junction of the isolated eyes in Kesselyak's saline. Then
the iris ring was divided into six sectors as shown in Fig. 1. The dorsal (D)
sector and the vental three (VN, VT and V) sectors were incubated in the NG
solution (100/^g/ml) at a constant temperature of 20 °C for an appropriate time,
washed with Kesselyak's saline, and then implanted into the lentectomized host
eyes.
Histological examinations. Animals to be examined histologically were sacrificed at various time intervals after an administration of NG or the implantation
of the iris pieces. For the fixed eyes sections were cut at 10 /an through the lens
regenerates with the usual paraffin techniques and were stained by Mallory's
staining method.
RESULTS
1. Supernumerary lens formation from the ventral half of the iris
Three series of experiments were achieved from 1970 to 1972 (Table 1). In
two series, A and C, the NG crystal was administered immediately after lentectomy. For series B an administration of NG was performed 5 days after
lentectomy. If NG is incorporated into the DNA of iris cells, its effect will be
most pronounced by administration in the period of most active DNA synthesis,
which is around 5 days after lentectomy (Eguchi & Shingai, 1971). This trial,
Lens formation by a carcinogen
65
Fig. 1. A schematic drawing showing sectors of iris to be separated into pieces as
implants. D, Dorsal; N, nasal, T, temporal; and V, ventral.
however, resulted in no significant differences among the experimental series
(cf. Table 1).
The cornea became opaque within 5 days after an administration of NG, but
in many cases it gradually recovered its transparency.
Though depigmentation of the mid-dorsal iris epithelium was delayed more
or less in many cases, the external and histological observation indicated that
lens regeneration took place in all cases in the same manner as in the case of
simple lentectomy. In the animals which were included in accompanying tables
as unrecorded cases, eyes were so severely affected with NG as to render any
external examination impossible. However, even in these animals lens regeneration took place, as confirmed by histological preparations.
Supernumerary lens regeneration from the lateral or the ventral iris epithelium
was found in considerable numbers of experiments through all series (Table 1,
see Fig. 2). In one case, B-19 (cf. Table 1), three lenses were formed altogether;
two additional lenses regenerated, one from the dorso-nasal (DN) and one from
ventro-nasal (VN) portions of the iris epithelium, besides the one from the
dorsal portion. Depigmentation of the iris epithelium was often observed in the
ventral or lateral marginal portions of the iris in addition to the usual dorsal
portion. The depigmented parts other than the dorsal iris, however, did not
always form a lens rudiment. In the case in which the supernumerary lens
regeneration took place, a small lens vesicle was formed from a restricted area
of the depigmented part of the lateral or the ventral iris, and, moreover, repigmentation of once-depigmented cells was often observed.
The results as a whole indicated that the iris epithelial cells of all portions
around the pupil become capable of transformation into lens cells as a result of
NG administration.
2. The secondary removal of Jens regenerates {secondary lentectomy
from eyes with the supernumerary lens regeneration)
In this series of experiments the lens regenerates were removed again in order
to test the stability of the induced competence of the ventro-lateral iris epithelial
5
EMB
30
66
G. EGUCHI AND K. WATANABE
Table 1. Effect of N-Methyl-N'-nitro-N-nitrosoguanidine (NG) on the
regenerative transformation of the newt iris epithelium into lens
The position of the secondary lens regeneration is indicated by a diagram for each
individual case. The lens regenerates are shown with open circles. Experimental
numbers are indicated to the upper left of each diagram (A-3, B-6, and so on).
Experimental series
Time of NG administration
after lentectomy
No. of eyes operated
No. of available cases
examined
Lens regeneration from
dorsal iris alone
Supernumerary lenses
formed from both dorsal
and other portions of
iris epithelium
A-3
A
(Jan. 1970)
B
(Feb. 1970)
C
(Mar. 1972)
Immediately
5 days
Immediately
30
30
68
27
23
49
25
19
45
2
4
4
B-6
N
N T-
Details of each case with
supernumerary lens
regeneration induced
byNG
C-l
D
D
B-I2*
-N
B-19*
C-4
-N
D
T -
C-l 8"
D
T
B-27*
T
D
T-
D
N
C-2V
D
N
N
* Used for the experiment of secondary lentectomy. D, dorsal; N, nasal; T, temporal; V, ventral.
Lens formation
by a carcinogen
67
cells for transformation into lens cells. For this purpose, five animals (B-12,
B-19, B-27, C-18 and C-21, listed in Table 1) were used.
Series B. By the time of the secondary lentectomy (12 months after the primary lentectomy), the regenerated lenses in B-19 and B-27 were fused into a single
large lens. In B-12, one dorsal and a ventro-nasal (VN) small lens were well
separated. The recovery of iris was complete in these cases. Around 10 days
after the secondary lentectomy the depigmentation of the iris epithelium started
in two portions of the marginal iris; the mid-dorsal portions and the same
position where the additional lens had been regenerated about 12 months
previously (Fig. 3).
Series C. In each of two cases of this series, C-18 and C-21, two lens regenerates, from dorsal (D) and ventro-temporal (VT) portions, had not yet become
detached from the iris epithelium at the time of the secondary lentectomy 45
days after the primary lentectomy. After lentectomy with particular care a
small number of depigmented cells still remained on the pigmented epithelium
after the operation. Within 4 days of the secondary lentectomy depigmentation
began to extend in the pigmented epithelium around the portions to which the
primary lens regenerates had been connected. The lens vesicles were developed
from the depigmented parts, and finally two secondary lens regenerates with the
same size as the primary ones respectively were differentiated from the same
positions where the two primary lenses had been situated (Fig. 4 A, B, C).
The results obviously indicate that the induced competence of the iris
epithelial cells for transformation into lens cells through an administration of
NG has been stably maintained for at least 12 months.
3. Implantation of iris pieces treated with NG solution
Following a number of preliminary tests, treatment of the iris pieces with 100
//g/ml NG for 60 min was chosen as optimal for this series of experiments.
Four sectors (D, VN, YT and V, shown in Fig. 1) from the isolated iris rings
were respectively tested (Table 2). After the treatment a considerable number of
implants were found to have disintegrated by the time of fixation at 25 days
after implantation. Seventeen of 19 available dorsal implants (D) differentiated
into lens. As to the pieces of the ventral half of the iris, lens formation was
found in five implants (Fig. 5). In the control implants which were immersed in
saline before implantation, lens formation occurred only from the dorsal sector
(Table 2).
5-2
68
4B
G. EGUCHI AND K. WATANABE
69
Lens formation by a carcinogen
Table 2. Effects of N-methyl-N'-nitro-N-nitrosoguanidine (NG) on isolated iris
pieces implanted into lentectomized host eyes
(Symbols, D, VN, VT, and V, in the first column correspond to those in Fig. 1.)
Sectors of iris
_
-
-A.
Treatment
Dorsal
D
Ventral
VN
NG
Saline
NG
Saline
VT
NG
V
NG
Saline
Saline
Total
no. of
implants
Transformed
into lens
Lens
vesicle
Implants
remained
unchanged*
Implants
disintegrated
25
25
20
20
20
20
20
20
15
19
1
0
3
0
1
0
2
3
1
0
0
0
1
0
2
0
13
19
10
17
13
18
6
3
5
1
7
3
5
2
* Some implants were partially depigmented.
DISCUSSION
In the present experiment NG, a potent mutagenic as well as carcinogenic
compound, was administered at rather high dosage into lentectomized newt
eyes. Although severe toxic effects were brought about in a considerable
FIGURES
2-5
Fig. 2. A histological section of a typical case, B-6, in which a supernumerary lens
was regenerated from the ventro-nasal (VN) portion of the iris in addition to the
dorsal lens regeneration. This case was administered with a piece of NG crystal 5
days after lentectomy and was fixed 35 days thereafter. One large lens {DL) was
formed from mid-dorsal iris, the other {VNL) from the ventro-nasal. x 90.
Fig. 3. Regeneration of two lenses after secondary lentectomy at 12 months after the
primary lentectomy in B-12. A photograph inserted in the right upper corner shows
the inside view of the iris ring with two lens regenerates. This case was fixed 20 days
after removal of the primarily regenerated lenses. A section showing the regeneration of two lenses; one {DL) developed from the dorsal (D) iris and the other (VNL),
from the ventro-nasal (VN) iris, x 90.
Fig. 4. Regeneration of two lenses after secondary lentectomy at 45 days after the
primary lentectomy in C-18. (A) A sketch of the eye at the time of the secondary
lentectomy. (B) A sketch of the fixed (16 days after the secondary lentectomy) eye
cleared in methylbenzoate. Each of two lens regenerates {DL, dorsal, and VTL,
ventro-temporal) is formed at the same position of the iris epithelium from which
the primary lens regeneration had taken place. (C) A histological section through
well-differentiated lens regenerates, x 40. A photograph inserted in the right lower
corner shows another section through the ventro-temporal lens regenerate {VTL) at
the site where the regenerate is continuous with the iris epithelium, m, Mitotic
figure (x 150).
Fig. 5. A lens formed from the ventral iris sector (V) which was treated with NG
solution before the implantation, x 70.
70
G. EGUCHI AND K. WATANABE
number of the experiments, no decisive carcinogenetic processes were found in
the ocular tissues except hyperplasia of the cornea. It is a well-established fact
that Wolffian lens regeneration in newts occurs only from the dorsal sector of
the iris epithelium. This has been quite extensively confirmed through a number
of investigations in the century since the discovery of this phenomenon in
European, American and Japanese newts. In parallel with the present experiment, a simple lentectomy was made in about 1000 eyes of Triturus pyrrhogaster
obtained together with the materials used for the experiment of NG administration. No spontaneous supernumerary lens regeneration ever occurred.
The results of the experiment suggests strongly that NG acts on the ventral
half of the iris to alter it to be competent to transform into the lens without
exhibiting carcinogenic effects. The effect of NG can be interpreted as more or
less specific, not merely as a trigger. The fact that the induced competence of the
ventral half of the iris by NG is stably maintained was well demonstrated
through a secondary lentectomy from the eye with a supernumerary lens.
The effect of NG on the iris epithelial cells was also confirmed by means of
implantation of isolated iris pieces treated in NG solution immediately before
implantation. A considerable number of implants derived from ventral iris
differentiated into a typical lens with normal polarity. This suggests that NG is
incorporated by the iris epithelial cells and it induces them to transform into
lens cells.
A direct modification of DNA, such as by alkylation, is thought to be the
means by which NG acts as a mutagen or carcinogen. The possibilities of modification of DNA by this compound have been demonstrated by in vitro and in
vivo studies (McCalla, 1968; Craddock, 1968; Lawley, 1968; Sugimura et al.
1969; Nagao et al. 1969). In the present experiment also, it is quite possible that
the DNA of some iris epithelial cells incapable of transformation can be modified by the action of NG to become capable of transformation, because the
induced competence of these cells for transformation into lens is stable for at
least as long as 12 months.
However, in the present demonstration the ventral iris cells transformed
only into a specific pathway; that is, into lens cells, and never into any other
cell types. Furthermore, the iris epithelium conserves this specific metaplastic
differentiative capacity. It therefore seems likely that the iris epithelial cells are
already potentially canalized strictly towards the cell types of the lens. This
appears to be a far more restricted change than that observed by Sugimura,
Fujimura & Bab a (1970), who have demonstrated that various kinds of tissues
were formed, together with tumours, in the stomachs of mice which were recipients
of NG solution. It is difficult to interpret the specific transformation of cell types
in the present experiment by means of a non-specific action of NG on DNA.
The molecular analysis of the mode of action of NG in the present system
can be expected to provide useful information for accessing the mechanism of
cellular differentiation at molecular level in the higher organisms in general.
Lens formation by a carcinogen
71
The authors wish to express their cordial gratitude to Professor T. S. Okada for his
valuable advice in planning the present study and kind help in revising the manuscript, and
to Professor T. Sugimura, National Institute for Cancer Research, Tokyo, who kindly gave
us the NG used in the present study. They also thank Mr M. Okamoto for his kind help in
preparation of the text figures, and Miss H. Takada who took trouble in preparing the
manuscript.
G. Eguchi expresses his gratitude to Mrs Ruth M. Clayton for her critical reading of the
manuscript and valuable discussions during his stay in the Institute of Animal Genetics,
University of Edinburgh, and also to Mr A. Gillies for his kind help in revising the illustrations.
The present work was supported by the Grants for Basic Cancer Research nos. 9115 and
92112 from the Japan Ministry of Education.
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CRADDOCK,
(Received 23 November 1972)