In vitro development of the regenerating lens

DEVELOPMENTAL

In Vitro

BIOLOGY

19,

368-379 (1969)

Development

of the Regenerating

SARA EISENBERG-ZALIK Department

of Zoology,

University

AND VIOLET

of Alberta,

Lens’

SCOTT

Edmonton,

Alberta,

Canada

Accepted December 21, 1968 INTRODUCTION

In the newt the pigment cells of the dorsal iris have the unique capacity of regenerating a new lens after the original has been surgically removed. Many aspects of lens regeneration have been studied in vivo, and these were reviewed recently by Stone (1960), Reyer (1954,1962), and Yamada (1966, 1967a,b). After lentectomy the cells of the dorsal iris extrude their pigment granules (Eguchi, 1963, 1964; Karasaki 1964), synthesize RNA, protein, and DNA (Yamada and Karasaki, 1963; Yamada and Takata, 1963; Eisenberg and Yamada, 1966), go through a number of cell divisions (Eisenberg-Zalik and Yamada, 1967), and finally elongate and synthesize lens-specific proteins (Takata et al., 1964, 1966). Previously we reported in vitro culture of the dorsal iris undergoing lens regeneration (Eisenberg-Zalik and Meza, 1968). This paper extends our preliminary study and deals with the developmental capacities of the dorsal iris placed in culture at various stages of lens regeneration. Under the culture conditions used, it was found that once the lens placode forms (stage IV onward), some cells have the capacity to develop into lens fibers. MATERIAL

AND

METHODS

Adult newts, Triturus viridescens, were lentectomized as described by by Eisenberg and Yamada (1966). The lens was removed from both eyes, and at the desired regeneration time the regenerate of one eye was placed in culture while the other was fixed and used as a control. i ? T 3 7 ;* Dissection of the regenerates. One day prior to the experiments animals were placed in chlorine free water containing a penicillin-streptomycin mixture at a concentration of 200 units/ml (Microbiological Associates, Bethesda, Maryland). Newts were anesthetized in MS-222 (Sandox), and one eye was fixed in Bouin’s fixative while the other was used as the 1 This work Canada.

was supported

by a grant

from the National

368

Research

Council

of

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LENS

source of the regenerate. Lens regenerates were removed under aseptic conditions. The cornea1 incision which had been performed for lentectomy purposes was reopened with a sharp scalpel blade (Fig. 1, step S), and extended to the lateral and median extremes of the eye (Fig. 1, step 6). With the aid of iris scissors this incision was continued upward and circumferentially along the upper margin of the eye until it joined the

@

8

6

7

CORNEA

AND

DORSAL IRIS

/ 9 FIG. 1. Schematic represent’at’ion of culture technique and dissection procedure. Steps 1-4 and9, culture methods; 5-8, dissection of t’he regenerate; arrows indicate direction of the incision. For explanation see text.

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initial cornea1 incision (Fig. 1, step 7). The cornea with the attached dorsal iris was removed and placed in culture medium, where the dorsal iris was gently separated from the cornea1 tissue with the aid of two scalpel blades (Fig. 1, step S). Care was taken to obtain the dorsal iris free of other ocular tissues; it was then placed on agar blocks and cultured (Fig. 1, step 9). Culture methods. Agar blocks were prepared by mixing a 4 % aqueous agar solution and culture medium in a proportion 1: 1 (v/v) (Fig. 1, step 1). The culture medium used was medium 199 with 15 % fetal bovine serum (Microbiological Associates, Bethesda, Maryland). Penicillin and streptomycin were incorporated into the medium each at a concentration of 100 units/ml. The agar blocks were prepared and transferred to a sterile petri dish, which was then filled with a small amount of culture medium (Fig. 1, step 4). A detailed description of this method has been given previously (Eisenberg-Zalik and Meza, 1968). Tissues were incubated at 25”C, 5 % COe, and S5 % relative humidity. Histological methods. After the desired incubation period, the explants were fixed as follows. Several drops of 4 % melted agar were placed on top of the agar block containing the regenerate. After the former solidified, the tissue so enclosed was fixed in Bouin’s fixative. Explants as well as control eyes were dehydrated, embedded, sectioned, and stained with hematoxylin and eosin. Five-, lo-, 15-, and 20-day-old regenerates were studied. Five- and loday regenerates were cultured for 5, 10, and 15 days, and 15- and 20-day regenerates were cultured for 5 and 15 days. A total of 94 regenerates were studied; 9 or 10 regenerates per group. The criteria proposed by Yamada (1967a) were used to determine the developmental stages of the control regenerates. These are based on the system originally proposed by Sato (1940) for T. pyrrhogaster and adapted for T. viridescens by Stone and Steinitz (1953) and Reyer (1954). RESULTS

The use of agar as a base for the explants provided a solid support where cell migration from the iris was minimal. In all regenerates cell migration was almost nil even after 15 days in culture. The criteria used to determine whether cell activation continued were the presence of nuclear enlargement with the appearance of nucleoli, the occurrence of depigmentation, cell division, cell elongation, and formation of lens fibers. Throughout this study the explants were compared with the corresponding control regenerate to ascertain whether development continued.

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Five-Day Regenerates Controls for this series were classified as being between stages I and II. In this period activation of the pigmented dorsal iris cells is evidenced by nuclear enlargment and reduction of pigment. The interlaminar space between inner and outer epithelial layers of the dorsal iris becomes conspicuous (Fig. 2). The regenerates cultured for 5 days did not show any significant advancement in regeneration as compared with the controls. In all cases however, depigmentation continued; pigment granules appeared to be extruded from the iris cells directly into the surrounding agar. After 10 days in culture, the signs of nuclear activation became evident in these cells; some cells had two or three prominent nucleoli. The majority of the regenerates showed depigmentation and slight elongation, cells ranging from cuboidal to cylindrical. Furthermore, many

2. Representative control stage for the 5 day cultllred regenerates. Cleft inner and outer layers of t.he dorsal iris is evident. X180. FIG. 3. Five-day lens regenerate cultured for 10 days. Agar substrate is at the bottom of the photograph. Cells are partially depigment,ed. Pigment granules and nuclei have migrated to the base of the cell. X180. regenerate cultured for 15 days. Cells are completely depigFIG. 4. Five-day mented, and slight cell elongatjion has occurred. X288. FIG.

between

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cells had elongated without their being completely depigmented. In these cells nuclei migrated to the base of the cell, together with the pigment granules (Fig. 3). The latter appeared to surround the former. A consequence of the migration of pigment granules to the base of the cells was that the apical end appeared to be free of pigment. These cells had a ruffled free border similar to the cells in active pinocytosis. Mitotic figures were present in pigmented and depigmented cells though they were few in number. After 15 days in culture some explants developed a depigmented cell population, resembling that of stage IV regenerates (Fig. 4). Mitoses were not present after this period of culture. Ten-Day Regeneraks Controls for this series ranged between stages early IV to early V. The cleft between internal and external iris layers has enlarged, and a number of depigmented cells are present in the inner wall of the dorsal iris. The latter may become pseudostratified due to an accumulation of cells at all stages of depigmentation (Fig. 5). A distinctive characteristic of regenerates at these stages was the very high degree of cell proliferation occurring in explants cultured for 5 days. Mitotic figures were abundant and occurred in pigmented and depigmented cells. The general appearance of these explants was that of a mass of depigmented cells undergoing mitosis (Fig. 6). This behavior continued in regenerates cultured for 10 days. In this series one explant showed structures resembling primary lens fibers of the core region of a stage VIII regenerate (Fig. 7). After 15 days in culture, most of the cells present in the explants were depigmented but a few remained full of pigment granules. The cells did not elongate or form fibers in the majority of the explants (Fig. 8). Mitotic figures had disappeared by the fifteenth day in culture. Fifteen-Day Regenerates Control regeneration stages for this series were between VI and VII. Depigmented cells of the inner layer of the lens vesicle stop dividing and start to elongate into primary lens fibers. The presumptive lens epithelium and the lens stalk become evident, and these are the sites where the mitotic cell population of the regenerating lens is located (Fig. 9). After 5 days in culture the regenerates showed a number of cells which had elongated into primary lens fibers, developing a structure resembling the lens fiber core of stages IX to X (Fig. 10). The proliferating cell population originally located in the lens stalk and presumptive lens epithelium was usually present at the periphery of the

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FIG. 5. Representative control for the lo-day cultured regenerates. A number of depigmented cells are present in the inner wall of the dorsal iris. X180. FIG. 6. Regenerate, 10 days after lentectomy cultured for 10 days. A large number of depigmented and partially depigmented cells are present. Mitotic figlu+es are abundant in other sections of this regenerate. X180. FIG. 7. Another regenerate of the same series as that of Fig. 6. A core region similar to that of a stage VIII regenerate has developed. Mitotic figures are abundant in the peripheral regions of the explant. X180. FIG. 8. Ten-day regenerate cultured for 15 days. An abundant depigmented cell population is present, but ILO lens fiber formation has occurred. X180.

explant, where it had given rise to an abundant population of depigmented cells. This was evident from the frequent mitotic figures present in this area. After 15 days in culture many of these regenerates had differentiated fibers corresponding to approximately stage X (Fig. 11). However no lens epithelium was present as such in any of the cu1tures.A few cases showed a population of depigmented cells at the periphery of the explants. These cells had not elongated. Twenty-Day Regenerates Control regenerates for this series ranged from stages IX to X. The primary lens fiber core is almost completely developed, and secondary lens fiber formation is taking place. The presumptive lens epithelium varies from one to two cell layers in thickness, and it is still continuous with the cells of the lens stalk (Fig. 12). The 20-day regenerates cultured

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FIG. 9. Representative control stage for the &day cultured regenerates. Depigmented cells of the inner layer of the lens vesicle are elongating into primary leus fibers. X180. FIG. 10. Fifteen-day regenerate after 5 days in culture. Lens fibers have differentiated in this explant. X180. FIG. 11. Regenerate, 15 days after lentectomy cultured for 15 days. A lens fiber core is present, but no lens epithelium has developed. X 180. FIG. 12. Control for the 20.day cultured regenerates. The primary lens fiber core has developed and secondary lens fibers are differentiating. X72. FIG. 13. Twenty-day regenerate cultured for 5 days. Secondary lens fibers have continued differentiating. The lens epithelium is flattening to become squamous in type. X180. FIG. 14. Twenty-day regenerate cultured for 15 days. Lens fibers have oriented around secondary centers of elongation. No lens epithelium is present. X72.

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for a j-day period continued to differentiate and attained stages XI to XII. Rlitotic figures were present in pigmented as well as depigmented cells, but these cells did not elongate. The lens epithelium either flattened into a squamous type cell, or elongated into fibers (Fig. 13). Secondary lens fibers continued to differentiate; in many cases they oriented around a secondary center of elongation which was established perpendicularly to the original primary lens fiber core (Fig. 14). After 15 days in culture these regenerates developed into lentoids in lvhich the development of primary and secondary lens fibers correspond to that of a mature lens. These regenerates, however, did not undergo a harmonious differentiation, but were flattened and had an elipsoidal shape. Lens epithelium was not present as such in any of the explants. From the foregoing experiments, development of the lens regenerates under the i?l vitro conditions employed may be summarized as follow. Once nuclear activation appears and reduction of pigment granules has started in the dorsal iris (stages I, II) the cells continue to undergo nuclear activation and depigmentation. They give rise to a number of depigmented cells capable of elongating into a cuboidal or cylindrical shape. The regenerates can proceed to stage III with extensive depigmentation, but they are not capable of forming lens fibers. After depigmentation is accomplished in some cells (stages III to IV), the cells are capable of entering mitosis and proliferating. These cells give rise to a depigmented cell population that ceases to divide after 1.5 days in culture. They remain depigmented and are not capable of differentiating into lens fibers in the majority of cases. After depigmentation is completed and ccl1 elongation begins (stages VI to VII), some fibers differentiate under culture conditions. When primary lens fibers have developed and secondary lens fibers have started to differentiate (stages IX to X), regenerates develop in vitro into structures resembling adult lenses. In these cases lens epithelial cells do not remain as a dividing proliferating population, but elongate and form lens fibers. Secondary lens fibers do not develop around the original primary lens fiber core, but orient around secondary nuclei of elongation.

There is limited information regarding in vitro development of lens regenerates although a number of investigators are engaged in the study of this phenomenon. The first attempt to culture the dorsal iris in vitro was that of Stone and Gallagher (19.33). The normal iris membrane of nonlentectomized newts was cultured on pieces of rayon acetate in a

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medium composed of horse serum and chick embryo extract. The authors reported absence of lens regeneration after 24 days in culture. A few iris membranes, however, were capable of giving rise to lens tissue when transplanted into a lentectomized eye. More recently Eguchi (1967), working with T. pyrrhogaster, cultured the iris membrane and the iridocorneal complex on solid media composed of agar, Tyrode solution, chick embryo extract, and horse serum. He succeeded in culturing the regenerating lens only as part of the iridocorneal complex. Cultures of the isolated iris ring showed suppression of lens regeneration, followed by disintegration of the iris tissue. Eisenberg-Zalik and Meza (1968) reported that maintenance of a CO2 atmosphere was essential for the optimal development of regenerates. It may be that the absence of regeneration in the isolated dorsal iris (Eguchi, 1967) was due to lack of CO2 atmosphere. In general our findings, based on the culture of isolated dorsal iris, agree in several aspects with those of Eguchi (1967), based on culturing the lens as part of the iridocorneal complex, and with the preliminary results of Reese as reported by Yamada (1967a). After nuclear activation has occurred (stages I to II), depigmentation continues. Following depigmentation (stages III to IV), the cells are capable of proliferating i?z vitro but remain undifferentiated. After cells start elongating (stages V and onward), further progress of differentiation can be obtained in vitro. From the results obtained in this study one can distinguish three stages in the transformation of iris cells into lens cells: depigmentation, multiplication, and elongation. Whether specific factors in the ocular environment responsible for inducing each of these stages can be reproduced in vitro remains to be established. In this context some preliminary results obtained by these authors seem to be of interest. Ten-day regenerates which give rise to a depigmented cell population at 5 % CO2 atmosphere, differentiate primary lens fibers at higher CO2 concentrations. A similar situation has been reported by Morton (1967) for erythrocyte maturation where higher CO* concentrations, up to 30%, stimulated reticulocyte and erythrocyte formation from immature marrow cells. This situation suggests that factors simulating conditions for cell differentiation may be developed in in vitro systems. The observation that, once depigmented cells begin to elongate, they can continue fiber formation in vitro is significant. A similar finding based on in. vivo studies was reported by Ikeda (1936) and Ikeda and Amatatu (1941). They found that the regenerating lens continued its development in the fourth brain ventricle of Hynobius larva, only if it was transplanted after the lens vesicle stage.

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Appearance of tissue-specific proteins is first detected by the fluorescent antibody technique in the elongated cells of the inner cell layer of stage IV regenerates (Takata et al., 1964). It appears that once cells stop synthesizing DSA (Eisenberg and Tamada, 1966) and start producing lens-specific proteins, they are capable of differentiating a lens fiber ilz vitro. A long-lived messenger RKA is synthesized in the developing lens of the chick embryo, (Reeder and Bell; Scott and Bell 1963); and the bovine lens epithelium (Papaconstantinou, 1967; Stewart and I’apaconstantinou 1967). Whether a similar situation exists in the regenerating lens remains to be established.

The development in vitro of the regenerating lens at successive time intervals after lens removal was studied in the adult newt Triturus uihdescens. Regenerates were cultured using an agar substrate in which a synthetic medium with serum supplement was incorporated. Explants were incubated at 25”C, 5 % CO2, and 85% relatively humidity, and cultured for 5, 10, and 15 days. Regeneration stages I to II, in which nuclear activation and depigmentation are starting to occur, continue to extrude their pigment granules and give rise to a population of completely depigmented cells. After depigmentation has occurred in vivo in some cells of the dorsal iris (stages III to IV), a depigmented cell population is produced capable of limited proliferation but incapable of lens fiber differentiation. When cell elongation has occurred in the depigmented cells of the inner wall of the regenerating lens (stages V and onward), explants are capable of developing lens fibers in vitro. These results are discussed and related to the available information on ILKA and protein synthesis in the lens rcgencrating system. REFP:Ilectron microscopic stildies 011 1e11sregeneratiolr. I. Mechallisms of depigmentation of the iris. Embrl/ologia 8, 45-02. EGUCHI, CT. (1964). Elect.ron microscopic st,lldies on leus regeneration. II. Formation and growth of the lells vesicle a11d differentiation of lens fibers. E’nzbqo2oyia 8, 247-287. I~k;LVcblI, (:. (1967). In vilro alkalysis of Wolfliau lerls regelxratioll. I)iRerentiation of the regenerating lens rlldimellt of the lIeat, Y’f,il~rrr~s pyrrhogaslrr. ZC’mDryologiu 9, 24-2(X. I~rSEKrrERC:, s., alld YAMADA, T. (19M). A stltdy of l>NA synthesis dtxing the transformation of iris into lens in the lentectomized newt. J. Excpll. Zoo!. 162, 353%3G8. EISENBERW%ALIK, S., alld ~IEZA, I. (19G8). In vitro caltltre of the regelleratirlg lens. *Ya/,cr.e 217, 179~180, EGUC'HI,

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EISENBERG-ZALIK, S., and YAMADA, T. (1967). The cell cycle during lens regeneration. J. Exptl. Zool. 166, 385-394. IKEDA, Y. (1936). Neue Versuche zur Analyse der Wolffschen Linsenregeneration. Arb. Anal. Inst. Kaiserl. Japan. Univ. Sendai 18, I-16. IKEDA, Y., and AMATATU, H. (1941). rber den Unterschied der Erhalturlgsmiiglichkeit der Linse bei zwei Urodelenarten (Triturus pyrrhogaster und Hynobius nebulosus), die sich beziiglich der F&higkeit zur Wolffschen Linsenregeneration voneinder wesentlich verschieden verhalten. Japan. J. Med. Sci. (I. Anal) 8, 205-226. KAIGASAKI, S. (1964). An electron microscopy strtdy of Wolffian lens regeneration in the adkIlt llewt. J. Ullrastruct. Bes. 11, 246-273. ,\IORTON, H. (1967). Role of carbon dioxide in erythropoiesis. Nuture 216, llGG1167. PAPACONSTANTINOU, J. (1967). Molecular aspects of lens cell differerrtiation. Science 166, 338-346. REEDER, R., and BELL, E. (1965). Short and long-lived messenger RNA in embryonic chick lens. Science 160, 71-72. REYER, 1~. W. (1954). Hegenerat#ion of the lens irr the amphibiarr eye. Quark. ZZev. Biol. 29, I-4G. REYE~, 11. W. (1962). Regerreratiou in the amphibian eye. 1n “Regeneration” (D. Rudnick, ed.), pp. 211-265. Ronald Press, New York. SATO, T. (1940). 1:ergleicheude Studier1 iiber die Geschwindigkeit der Wolffschen Linserrregeneration bei Triton taeniatus und bei L)iern@ilzLs pyrrhogasler. Arch. En&chlungsmech. Organ. 140, 570-G13. Scow, W. B., and BELL, K. (1965). Messenger RNA rrtilizatiou during development of chick embryo lens. Science 147,405-407. STEwART, J. A., and PAPACoNsTANTINoU, J. (1967). Stabilization of RNA templates in bovine lens epithelial cells. J. Mol. Biol. 29, 357-370. STONE, L. S. (1960). Regeneration of the lens, iris and neural retina in a vertebrate eye. Yale. J. Biol. Med. 32,464-473. STONE, L. S., and GALLAGHEIL, S. B. (1958). Lens regeneration restored to iris membranes when grafted to neural retina environment after cultivation in vitro. J. Exptl. Zool. 139, 247-261. STONE, L. S., and STEINITz, H. (1953). Regeneration of lenses in eyes with intact aud regenerating retina in adult Y’riturus viridescens. J. Exptl. Zool. 124, 435468. TAKATA, C., ALURIGHT, J. F., and YAMADA, T. (1904). Lens antigens in a lens regeneating system studied by the immunofluorescent technique. Develop. Biol. 9,386397. in TAKATA, C., ALBRIGHT, J. F., and YAMADA, T. (1966). Gamma crystallins Wolffian lens regeneration demonstrated by immunofluorescence. Develop. Biol. 14, 382-400. YAMADA, T. (1966). Control of tissue specificity. The patter-u of cellular syuthetic activities in tissue transformation. Am. Zoologist 6, 21-31. YAMADA, T. (1967a). Celhrlar and subcellular events in Wolffian lens regeneration. In “Current Topics in Developmental Biology” (A. Monroy and A.A. Moscona, eds.), 1’01. 2, pp. 247-283. Academic Press, New York. YAMADA, T. (1967b). Cellular synthetic activities in induction OF tissue trans-

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formation. Ciba Found. S!/mp. Cell Di$erentialion pp. llG126. Little, Brown, Boston, Massachusetts. Y.\MAUA, T., and KABASAKI, S. (1963). Nnclear RNA synthesis in newt iris cells engaged in regenerat)ive t,ransformation into lens cells. Develop. Riol. 7,595~604. study of protein synYAMADA, T., and TAKATA, C. (1963). An autoradiographic thesis in regenerative tissue transformation of iris into lens in the newt. Deoelop. Rio/. 8, 358-369.