/ . Embryo!, e.xp. Morpli. Vol. 30, 1, pp. 63-71, 1973 6 3

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Elicitation of lens formationfrom the 'ventral iris' epithelium of the newt by acarcinogen, iV-methyl-iV'-nitro-iV-nitrosoguanidine

By GORO EGUCHP AND KENJI WATANABE1

From the Laboratory for Differentiation and Morphogenesis,Institute of Biophysics, University of Kyoto

SUMMARYA potent carcinogenic compound, iV-methyl-./V'-nitro-./V-nitrosoguanidine (NG), was

administered to the lentectomized eyes of the newt, Triturus pyrrhogaster. Supernumerarylenses were formed from the dorsal iris and even from the ventral iris, which never transformsinto lens following simple surgical lentectomy or the grafting of isolated iris pieces into len-tectomized eyes. After the administration of NG the pigmented epithelial cells of theventral iris were induced to gain the competence for transformation into lens cells. Theinduced 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 lentectomytook place at the same positions of the ventral area of the iris where the primary lens regenera-tion had been induced by NG.

The effects of NG were also confirmed by implanting isolated pieces of iris treated with NGsolution into lentectomized eyes. Five implants out of 42 available experiments on the ventralhalf of the iris were found to be transformed into lens.

INTRODUCTION

The iris epithelium of newts is capable of transforming into lens. When a lensis surgically removed from the eye, the lens regeneration occurs always onlyfrom the dorsal marginal portion of the iris epithelium. Among a large numberof papers so far published (for reviews see Reyer, 1954, 1962; Scheib, 1965;Yamada, 1967) there are none indicating the possibility of the transformationof ventral iris epithelium into lens. When isolated iris pieces are implanted inlentectomized host eyes, the pieces derived from the ventral half of the iris areincapable of transformation into a lens, but ones from the dorsal half are capableof transformation (Sato, 1930, 1951; Mikami, 1941). At the cellular level,Eguchi & Shingai (1971) have recently suggested that there are two different cellpopulations, one being capable and the other incapable of transforming intolens, and cells of the former type are densely localized in the dorsal part of theiris. 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 Bio-physics, 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 in-capable of transformation.

This paper deals with the finding that the administration of iV-methyl-iV'-nitro-JV-nitrosoguanidine (NG) to lentectomized newt eyes successfully elicitssupernumerary lenses from the ventral or the lateral portions of the iris epitheliumin 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 thepresent experiment. A small particle of NG was inserted into the lentectomizedeyes. In another series of experiments pieces of iris were treated in the NGsolution and were then implanted into the lentectomized eyes. The procedurefor lentectomy was the same as given in Eguchi & Shingai (1971). The lensremoval 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 placedinto the posterior eye chamber either immediately after or 5 days after thelentectomy. The inserted NG particle remained at least for 1 h within the eyechamber.

Implantation of iris pieces treated with NG. The iris ring was obtained bycutting the irido-corneal junction of the isolated eyes in Kesselyak's saline. Thenthe 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 NGsolution (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 hosteyes.

Histological examinations. Animals to be examined histologically were sacri-ficed at various time intervals after an administration of NG or the implantationof the iris pieces. For the fixed eyes sections were cut at 10 /an through the lensregenerates with the usual paraffin techniques and were stained by Mallory'sstaining 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). Intwo series, A and C, the NG crystal was administered immediately after lentec-tomy. For series B an administration of NG was performed 5 days afterlentectomy. If NG is incorporated into the DNA of iris cells, its effect will bemost 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 asimplants. 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, butin many cases it gradually recovered its transparency.

Though depigmentation of the mid-dorsal iris epithelium was delayed moreor less in many cases, the external and histological observation indicated thatlens regeneration took place in all cases in the same manner as in the case ofsimple lentectomy. In the animals which were included in accompanying tablesas unrecorded cases, eyes were so severely affected with NG as to render anyexternal examination impossible. However, even in these animals lens regenera-tion took place, as confirmed by histological preparations.

Supernumerary lens regeneration from the lateral or the ventral iris epitheliumwas 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 fromventro-nasal (VN) portions of the iris epithelium, besides the one from thedorsal portion. Depigmentation of the iris epithelium was often observed in theventral or lateral marginal portions of the iris in addition to the usual dorsalportion. The depigmented parts other than the dorsal iris, however, did notalways form a lens rudiment. In the case in which the supernumerary lensregeneration took place, a small lens vesicle was formed from a restricted areaof the depigmented part of the lateral or the ventral iris, and, moreover, repig-mentation of once-depigmented cells was often observed.

The results as a whole indicated that the iris epithelial cells of all portionsaround the pupil become capable of transformation into lens cells as a result ofNG administration.

2. The secondary removal of Jens regenerates {secondary lentectomyfrom eyes with the supernumerary lens regeneration)

In this series of experiments the lens regenerates were removed again in orderto test the stability of the induced competence of the ventro-lateral iris epithelial

5 E M B 30

66 G. EGUCHI AND K. WATANABE

Table 1. Effect of N-Methyl-N'-nitro-N-nitrosoguanidine (NG) on theregenerative transformation of the newt iris epithelium into lens

The position of the secondary lens regeneration is indicated by a diagram for eachindividual case. The lens regenerates are shown with open circles. Experimentalnumbers are indicated to the upper left of each diagram (A-3, B-6, and so on).

Experimental series

Time of NG administrationafter lentectomy

No. of eyes operatedNo. of available cases

examinedLens regeneration from

dorsal iris aloneSupernumerary lenses

formed from both dorsaland other portions ofiris epithelium

A(Jan. 1970)

Immediately

30

27

25

2

B(Feb. 1970)

5 days

30

23

19

4

C(Mar. 1972)

Immediately

68

49

45

4

A-3D

B-6

Details of each case withsupernumerary lensregeneration inducedb y N G

N T -

B-I2*

- N T -

B-19*

B-27*

DC-l

D

D

D

N T

C-4

- N T -

C-l 8"

T N

C-2V

N N

D

D

* 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 pri-mary lentectomy), the regenerated lenses in B-19 and B-27 were fused into a singlelarge lens. In B-12, one dorsal and a ventro-nasal (VN) small lens were wellseparated. The recovery of iris was complete in these cases. Around 10 daysafter the secondary lentectomy the depigmentation of the iris epithelium startedin two portions of the marginal iris; the mid-dorsal portions and the sameposition where the additional lens had been regenerated about 12 monthspreviously (Fig. 3).

Series C. In each of two cases of this series, C-18 and C-21, two lens regene-rates, from dorsal (D) and ventro-temporal (VT) portions, had not yet becomedetached from the iris epithelium at the time of the secondary lentectomy 45days after the primary lentectomy. After lentectomy with particular care asmall number of depigmented cells still remained on the pigmented epitheliumafter the operation. Within 4 days of the secondary lentectomy depigmentationbegan to extend in the pigmented epithelium around the portions to which theprimary lens regenerates had been connected. The lens vesicles were developedfrom the depigmented parts, and finally two secondary lens regenerates with thesame size as the primary ones respectively were differentiated from the samepositions where the two primary lenses had been situated (Fig. 4 A, B, C).

The results obviously indicate that the induced competence of the irisepithelial cells for transformation into lens cells through an administration ofNG 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 ringswere respectively tested (Table 2). After the treatment a considerable number ofimplants were found to have disintegrated by the time of fixation at 25 daysafter implantation. Seventeen of 19 available dorsal implants (D) differentiatedinto lens. As to the pieces of the ventral half of the iris, lens formation wasfound in five implants (Fig. 5). In the control implants which were immersed insaline before implantation, lens formation occurred only from the dorsal sector(Table 2).

5-2

68 G. EGUCHI AND K. WATANABE

4B

Lens formation by a carcinogen 69

Table 2. Effects of N-methyl-N'-nitro-N-nitrosoguanidine (NG) on isolated irispieces implanted into lentectomized host eyes

(Symbols, D, VN, VT, and V, in the first column correspond to those in Fig. 1.)

Implants Implantsremained dis-

unchanged* integratedSectors of iris_ - -A . Treatment

Totalno. of

implantsTransformed

into lensLens

vesicle

Dorsal

Ventral

D

VN

VT

V

NGSalineNGSalineNGSalineNGSaline

2525

2020

2020

2020

* Some implants were

1519

10

3010

partially

23

10

00

10

depigmented.

20

1319

1017

1318

63

51

73

52

DISCUSSION

In the present experiment NG, a potent mutagenic as well as carcinogeniccompound, was administered at rather high dosage into lentectomized newteyes. 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 lenswas regenerated from the ventro-nasal (VN) portion of the iris in addition to thedorsal lens regeneration. This case was administered with a piece of NG crystal 5days after lentectomy and was fixed 35 days thereafter. One large lens {DL) wasformed 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 theprimary lentectomy in B-12. A photograph inserted in the right upper corner showsthe inside view of the iris ring with two lens regenerates. This case was fixed 20 daysafter removal of the primarily regenerated lenses. A section showing the regenera-tion 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 theprimary lentectomy in C-18. (A) A sketch of the eye at the time of the secondarylentectomy. (B) A sketch of the fixed (16 days after the secondary lentectomy) eyecleared in methylbenzoate. Each of two lens regenerates {DL, dorsal, and VTL,ventro-temporal) is formed at the same position of the iris epithelium from whichthe primary lens regeneration had taken place. (C) A histological section throughwell-differentiated lens regenerates, x 40. A photograph inserted in the right lowercorner shows another section through the ventro-temporal lens regenerate {VTL) atthe site where the regenerate is continuous with the iris epithelium, m, Mitoticfigure (x 150).Fig. 5. A lens formed from the ventral iris sector (V) which was treated with NGsolution before the implantation, x 70.

70 G. EGUCHI AND K. WATANABE

number of the experiments, no decisive carcinogenetic processes were found inthe ocular tissues except hyperplasia of the cornea. It is a well-established factthat Wolffian lens regeneration in newts occurs only from the dorsal sector ofthe iris epithelium. This has been quite extensively confirmed through a numberof investigations in the century since the discovery of this phenomenon inEuropean, American and Japanese newts. In parallel with the present experi-ment, a simple lentectomy was made in about 1000 eyes of Triturus pyrrhogasterobtained together with the materials used for the experiment of NG administra-tion. No spontaneous supernumerary lens regeneration ever occurred.

The results of the experiment suggests strongly that NG acts on the ventralhalf of the iris to alter it to be competent to transform into the lens withoutexhibiting carcinogenic effects. The effect of NG can be interpreted as more orless specific, not merely as a trigger. The fact that the induced competence of theventral half of the iris by NG is stably maintained was well demonstratedthrough a secondary lentectomy from the eye with a supernumerary lens.

The effect of NG on the iris epithelial cells was also confirmed by means ofimplantation of isolated iris pieces treated in NG solution immediately beforeimplantation. A considerable number of implants derived from ventral irisdifferentiated into a typical lens with normal polarity. This suggests that NG isincorporated by the iris epithelial cells and it induces them to transform intolens cells.

A direct modification of DNA, such as by alkylation, is thought to be themeans by which NG acts as a mutagen or carcinogen. The possibilities of modi-fication of DNA by this compound have been demonstrated by in vitro and invivo studies (McCalla, 1968; Craddock, 1968; Lawley, 1968; Sugimura et al.1969; Nagao et al. 1969). In the present experiment also, it is quite possible thatthe DNA of some iris epithelial cells incapable of transformation can be modi-fied by the action of NG to become capable of transformation, because theinduced competence of these cells for transformation into lens is stable for atleast as long as 12 months.

However, in the present demonstration the ventral iris cells transformedonly into a specific pathway; that is, into lens cells, and never into any othercell types. Furthermore, the iris epithelium conserves this specific metaplasticdifferentiative capacity. It therefore seems likely that the iris epithelial cells arealready potentially canalized strictly towards the cell types of the lens. Thisappears to be a far more restricted change than that observed by Sugimura,Fujimura & Bab a (1970), who have demonstrated that various kinds of tissueswere formed, together with tumours, in the stomachs of mice which were recipientsof NG solution. It is difficult to interpret the specific transformation of cell typesin 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 systemcan be expected to provide useful information for accessing the mechanism ofcellular differentiation at molecular level in the higher organisms in general.

Lens formation by a carcinogen 71The 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, andto Professor T. Sugimura, National Institute for Cancer Research, Tokyo, who kindly gaveus the NG used in the present study. They also thank Mr M. Okamoto for his kind help inpreparation of the text figures, and Miss H. Takada who took trouble in preparing themanuscript.

G. Eguchi expresses his gratitude to Mrs Ruth M. Clayton for her critical reading of themanuscript 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 and92112 from the Japan Ministry of Education.

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in the newt iris epithelium. Dev. Growth, Diff. 13, 337-349.LAWLEY, P. D. (1968). Methylation of DNA by Af-Methyl-./V-nitroso-./V'-nitroguanidine.

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iV-nitroso-P-toluenesulfonamide with DNA in vitro. Biochem. biophys. Acta 155, 114-120.MIKAMI, Y. (1941). Experimental analysis of the Wolffian lens-regeneration in adult newt,

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REYER, R. W. (1954). Regeneration of the lens in the amphibian eye. Q. Rev. Biol. 29, 1-46.REYER, R. W. (1962). Regeneration in the amphibian eye. In Regeneration (ed. D. Rudnick),

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YAMADA, T. (1967). Cellular and subcellular events in Wolffian lens regeneration. In CurrentTopics in Developmental Biology, vol. 2 (ed. A. A. Moscona and A. Monroy), pp. 247-283.New York: Academic Press.

(Received 23 November 1972)