Secondary lens formation caused by implantation of pituitary into the eyes of the newt, Notophthalmus

DEVELOPMENTAL

Secondary

BIOLOGY

52, 126-140 (1976)

Lens Formation Caused by Implantation

of Pituitary into

the Eyes of the Newt, Notophthalmus JEANNE Smith

A.

College,

POWELL

AND

Northampton, Accepted

April

NEIL

Massachusetts

SEGIL' 01060

19,1976

Whole pituitary glands, as well as equivalent-sized pieces of kidney, liver, and adrenal tissue, were implanted into the anterior chamber of the adult newt eye. Secondary lens formation was present in 16 out of 17 cases of the pituitary implantation experiments in which the pituitary tissues were exposed to the anterior chamber fluid. In only one case out of 23, did another implanted tissue (liver) stimulated secondary lens formation. The extent of secondary lens formation was correlated with the position (closeness) of the implant in relationship to the dorsal iris. In all cases of secondary lens formation from the dorsal iris stimulated by tissue implants, the polarity of the developing lens was reversed so that the pole of the fiber axis was directed toward the implant rather than the retina. It appears that the ectopic pituitary produces a diffusible substance capable of initiating lens formation from the dorsal margin of the iris and capable of determining the orientation of the fiber axis pole of the developing lens. INTRODUCTION

Upon extirpation of the lens of the adult newt, Notophthalmus viridescens, a process begins which culminates in the replacement of the removed lens, a process that involves the transformation of epithelial cells of the dorsal iris into differentiated, crystalline lens cells (Reyer, 1954; Yamada and McDevitt, 1974). Although considerable information has accumulated concerning the molecular (Yamada, 1966, 1967a,b) and fine structural events (Eguchi, 1963, 1964; Karasaki, 1964; Dumont et al., 1970; Dumont and Yamada, 1972; Yamada and Dumont, 1972) accompanying the transformation of iris tissue and the redifferentiation process, very little is understood concerning the environmental factors controlling or releasing the process of cellular transformation. The transformation of iris into lens is normally dependent on the presence of an intact neural retina. If the neural retina is removed or damaged, lens regeneration is delayed until the visual retina tissue is regenerated by the pigmented retina I This research was supported in part by a DuPont research grant to the Department Sciences, Smith College. c opvght All

rights

0

of the Biological

1976 by Academic Press, Inc. of reproduction in any form reserved.

(Stone and Steinitz, 1953a; Stone 1958b; Reyer, 1971). If the iris is separated from, the neural retina by means of a pliofilm disc (Stone, 1958a), no transformation of the iris takes place. Furthermore, iris transformation in vitro is dependent upon the presence of neural retina (Yamada et al., 1973). The phenomenon of regeneration in many systems is dependent upon the presence of neural tissue, which is thought to produce a trophic factor (Singer, 1965) necessary for dedifferentiation and proliferation of the regenerating organ. For example, forelimb regeneration in the newt is dependent upon, among other factors, intact nervous tissue (Schotm, 1926; Singer, 1960). There is some evidence that the “neurotrophic” substance from one system may substitute for that of another. Iris cells transplanted into the milieu of regenerating nerves (the forelimb blastema) produce lens regenerates (Reyer et al., 1973). Also. brachial ganglia, taken from animals regenerating forelimbs and implanted into the anterior chamber of the eye, can support secondary lens formation (Powell and Powers, 1973). The pituitary may play an inducing role 128

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Secondary

similar in both lens and limb regeneration although its role may be more significant in the limb system. The action of the pituitary has been associated with the early events in both limb regeneration (Schotti and Hall, 1952; Thornton, 1968; Sate and Inoue , 1973) and lens regeneration (Schotti and Murphy, 1953; Stone and Steinitz, 1953b; Stone, 1957; Connelly et aZ., 1973). In limb regeneration the presence of the pituitary is necessary during the initiation stages of regeneration. When effective hypophysectomy precedes amputation, limb regeneration is inhibited (Hall and Schotti, 1951) while hypophysectomy 10 days or more after amputation has little or no effect on further regeneration (Schotti and Hall, 1952). More recently it has been shown that the effect of hypophysectomy preceding amputation is only one of delay in the regenerative process (Sato and Inoue, 1973), a delay which can be overcome by nutritional supplements (Tassava, 1960; Sam and Inoue, 1973) and administration of growth hormone (Wilkerson, 1963; Sato and Inoue, 1973). Hypophysectomy also causes a delay, or decrease in the rate or growth, in the process of lens regeneration (Schotm and Murphy, 1953; Stone and Steinitz, 1953b; Stone, 1957). Lag in lens regeneration following hypophysectomy has been interpreted as a response to a general metabolic effect rather than a direct result of specific action of (lack of hormone from) the pituitary (Schotte and Murphy, 1953). However, Zalokar (1944) has noted that implantation of the pituitary into larval (Triton cristatus) eyes, explanted in culture, causes far greater incidence of lens regeneration than that found in control eyes with no implants. Furthermore, when adult iris tissue is cultured in uitro, adult pituitary enhances lens regeneration (Connelly et al., 1973). We thought that dorsal iris might respond directly to local activity of pituitary tissue. The present set of experiments has

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been designed to test the ability of ectopic pituitary implants in the eye to support secondary lens formation in the presence of an intact lens. Lens regeneration is inhibited in the presence of a healthy host lens, but may occur if the original lens is disturbed and thus begins to degenerate (Takano et al., 1958). Regenerating retina (Williams, 1970; Williams and Higginbotham, 1975) and ganglia supporting limb regeneration (Powell and Powers, 1973) implanted into the anterior chamber of the eye can support inhibited attempts at secondary lens formation in the presence of an intact lens. Our present results show that pituitary implanted into the eye supports secondary lens formation to even more advanced stages than does either regenerating retina or spinal ganglia. MATERIALS

AND

METHODS

Adult newts, Notophthalmus uiridestens, purchased from Connecticut Valley Biological Supply Co., ranged in length from 70 to 80 mm and were reared in finger bowls containing pond water at 15°C. During this time they were fed bits of chicken liver weekly. Prior to operations, newts were anesthetized in a sterile solution of 1:lOOOtricaine methanesulfonate (Finquel, Ayerest Co.). All operations were performed on a piece of moistened gauze, under a dissecting microscope. Immediately following operations, the animals were placed in individual moist chambers (23-25°C). Pituitary glands were removed from donor newts and placed in Amphibians Ringer’s for a few minutes preceding the operation. The host newt was then prepared for the implant by making an antero-posterior cut in the cornea. With the aid of watchmaker’s forceps, the pituitary implant was then pushed through the corneal slit and positioned between the dorsal iris and the cornea. As controls, pieces of liver, pancreas, or adrenal tissue were inserted into the eyes contralateral to those receiving the pituitary implants; other

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DEVELOPMENTAL BIOLOGY

eyes were sham-operated or lentectomized. All implants were trimmed to the approximate size of a pituitary or, in the case of adrenal bodies, several pieces were used to approximate the amount of pituitary tissue inserted. The heads of the animals were fixed in Bouin’s fixative at intervals of 9,12,16,18, and 20 days following operations. The heads were decalcified in 5% nitric acid in 80% ethanol for 3 days, the solution being changed once each day, and were dehydrated and then cleared in toluene. The heads were embedded in paraffin and sectioned at 10 pm. Sections were stained with aldehyde fuchsin (Cameron and Steele, 1959).

VOLUME 52, 1976

posterior pituitary implant had a normal colloidal matrix consistency, as described by Copeland (19431,and stained positively for neurosecretory granules. Because of the number of implants growing for various periods of time, it was impossible to analyze the extent of granule loss due to time of implantation. There appeared to be no correlation between extent of neurosecretory granule staining and extent or presence of secondary lens formation. The analysis of the results of the pituitary implant operations showed that the position of the implants, in relation to the iris, varied in different cases. Placement of the pituitary (Table 1) was categorized in four ways: (1) intimate contact (cellular abutment) with the dorsal iris or secondRESULTS ary lens, (2) offset from the dorsal sector, Pituitary Implant Operations and Secondin the temporo-dorsal or naso-dorsal reary Lens Formation gion of the anterior chamber of the eye, or Secondary lens formation was found in directly lateral to the pupil, (3) wedged in the cornea, or (4) encapuslated in the cor16 out of 17 cases of the pituitary implantation experiments in which the pituitary nea. The analysis of the positional effect of was present in the eye and some of its the pituitary indicates that the inducing surface area was exposed to the anterior potential of the pituitary implant can be chamber fluid. In two other cases, one correlated with its placement (its distance with a pituitary implant (Fig. 1) and one from the iris and the amount of its surface with a liver implant, the lens had appar- area contiguous with the anterior chamber ently been damaged and was in the process fluid). of degeneration; early stages of secondary In some of the cases that exhibited optilens formation were detected in the dorsal mum conditions of pituitary placement iris of these eyes. Such regeneration has (contact between iris and implant), secbeen previously documented following lens ondary lens formation was as advanced as deterioration (Eguchi, 1961). In all other would be expected following simple lentecimplantation experiments the lens was in- tomy (Table 1, case nos. 26, 12, 16, and 17). tact and showed no signs of deterioration Since the assessment of placement was (all cases cited in Tables l-3) as indicated done following fixation, in cases where secby lack of vacuoles and presence of shat- ondary lens formation was far advanced tering of the hard crystalline lens material there was no way to judge whether the during sectioning (Figs. 4, B-10). Iris pituitary originally abutted the dorsal iris transformation therefore, when present, or whether the secondary lens grew into was attributed to the influence of the pitui- contact with the implant. In most cases in which the pituitary was found removed tary implant. from the dorsal sector (offset from dorsal Following fixation of the pituitary grown in the anterior chamber of the eye, iris or wedged in cornea; Table 11, lens there appeared to be a normal proportion formation was less advanced than might of anterior and posterior pituitary tissue be expected following lentectomy (Table 1, as compared to pituitary fixed in situ. The case nos. 9, 3, 5, 30, 18, 2, 7, and 14). In all

FIG. 1. Section through an eye in which a pituitary (P) had been implanted 18 days prior to sacrifice. The lens had apparently been damaged at the time of implantation and can be seen in this section as an irregular vacuolated tissue mass (L). Lens regeneration, as indicated by depigmentation of the dorsal iris (DI) is present. X 135. FIG. 2. Section through an eye in which a pituitary (P) had been implanted 20 days prior to fixation. An advanced (stage IX) secondary lens (SL) has formed from the dorsal iris in the presence of the primary lens (PL). Note that the polarity of the fiber axis is oriented toward the pituitary implant. X 135. FIG. 3. Enlargement of Fig. 2. The lens fiber forming cells (arrows) appear to originate from the outer lamina of the dorsal iris. x 225.

131

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DEVELOPMENTALBIOLOGY TABLE

VOLUME 52, 1976 1

PITUITARY IMPLANT EXPERIMENTS Case no.

Da s impTant grown in

eye 9 10 11

9 9 9

3 5 26” 30” 31”

12 12 12 12 12

12 18” 24 25

16 16 16 16

1 2 7 14 23

18 18 18 18 18

16 17” 29”

20 20 20

Proximity Intimate contact

of implant Offset from dorsal iris

to dorsal iris

Wedeed ” in cornea

Encaosulated in cornea

X

I III IV

X X X X

III III VII

X X

I IV

X

X X X X

VI III III No regeneration No regeneration IV IV No regeneration No regeneration

X X X

IX IX VI

X X X

o Contralateral eyes contained of secondary lens formation. h ARer Yamada, 1967b.

-

Stage of secondary lens formation

implants

of tissue other than pituatary

cases in which the pituitary was encapsulated in the cornea, no secondary lens formation was found (Table 1, case nos. 25, 1, and 23). Table 2 gives a summary of cases in Table 1, comparing stages of secondary lens formation with normal regenerative stages in the absence of lens. Two examples of cases classified in category 1 “intimate contact” (Tables 1 and 2) are shown in Figs. 2-5. The secondary lenses, in both cases, are in intimate contact with the implanted pituitary. Stages of regeneration are well advanced: stage IX after 20 days of implantation (Figs. 2 and 3) and stage VI after 16 days of implantation (Figs. 4 and 5). In the two cases in which secondary lens formation was well advanced (e.g., Figs. 2 and 3) internal lens

1Normal regenerative stage”

II-IV

IV-VI

VI-VIII

VII-X

IX-X

(See Table 2) and showed no signs

fibers appeared abnormally twisted and misshapen. Although there is no way to determine the cause of this improper development, in both cases it was noted that the pituitary implants were so closely apposed to the lens vesicle that the pituitary could have caused a mechanical disturbance. The second category shown in Table 1 “offset from dorsal iris” is recognized by analysis of serial sections of the eye and thus is difficult to illustrate. Secondary lens formation has taken place in cases in this category, although at a slower rate than those in category 1, and in general at a slower rate than would be anticipated following simple lentectomy (Table 2). The single case (no. 14, Table 1) which ex-

Secondary

POWELL AND SEGIL

veloping secondary lens 16 days postimplantation (Fig. 4), possesses a stage VI regenerate. In the one anomalous (in that regeneration was as advanced as would be expected following simple lentectomy) case in category 3, the pituitary implant presented a large surface area to the anterior chamber of the eye (Fig. 7) in comparison to the other implants in this category (e.g., Fig. 6). Finally, when the pituitary implant was found encapsulated in the cornea and presented no surface area to the anterior chamber, no secondary lens formation occurred. The eye depicted in Fig. 8 (case no. 23, Table 1) is typical of the three cases in category 4, “encapsulated in cornea.” In these eyes the pituitary implant was surrounded by cornea1 tissue and is excluded from the anterior chamber by the cornea1 basement lamella (Fig. 8).

hibited no lens regeneration was found in this category, but perhaps should be classed uniquely. In this eye the pituitary explant was found in cellular contact with lens tissue and was somewhat engulfed by lens epithelium. In the four cases in the third category, “wedged in cornea” (Table 11,some secondary lens formation occurred, but the progress of three of these cases (nos. 9, 18, and 7, Table 1) was retarded as compared to that in the preceding two categories. For example, Fig. 6 shows a stage III regenerate in an eye in which the pituitary was found wedged in the cornea following 16 days of implantation while an eye in which the pituitary was in contact with the deTABLE

2

PITUITARY Implant Experiments: COMPARISON OF SECONDARY LENS FORMATION WITH NORMAL LENS REGENERATION” Type of category

Intimate contact Offset from dorsal iris Wedged in cornea Encapsulated in cornea

Control Experiments

Number Secondary lens formaof cases tion compared to norin each ma1 lens regeneration category Not reReAbtarded tarded sent 6 i’

4 2

2 4

4 3

1

3

Control experiments were performed on eyes of several animals that received no pituitary tissue as well as on the contralatera1 eyes of animals with pituitary implants. This allows analysis of any possible systemic effects of the implanted pituitary on the contralateral eyes. The results of these control experiments are summarized in Table 3. Secondary lens formation did not occur in any of the control eyes in which other tissues (liver, pancreas, or adrenal) were implanted with the single ex-

1

3

’ This is a tabular summary of data from Table 1 to indicate the stages of secondary lens formation in eyes with pituitary implants compared to stages of normal lens regeneration following lentectomy. TABLE Tissue implant

Liver

Pancreas

Adrenal

133

Lens Formation

3

CONTROL EXPERIMENTS” Type of category Number Secondary lens formation compared to normal of cases lens regeneration in each category Not retarded Retarded Absent Intimate contact Offset from dorsal iris Wedged in cornea Intimate contact Offset from dorsal iris Wedged in cornea Intimate contact Wedged in cornea

’ Data taken from experiments

in which

2 2 6 2 1 3 6 1 implants

l(stage

III)

1 2 6 2 1 3 6 1

were grown in the eye for 12, 16, 18, and 20 days.

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DEVELOPMENTAL BIOLOGY

ception of one case of a liver implantation. In most cases of tissue implants, the dorsal iris was somewhat swollen but never was there any evidence of depigmentation or cleft formation between inner and outer lamina of the iris. In the single case of secondary lens formation among the controls (Fig. 9; Table 3) secondary lens formation was much retarded (stage III, 16 days following implantation) as compared to the contralateral eye of case no. 12 (Table 1) in which pituitary has caused secondary lens formation to stage VI (Figs. 4 and 5). It is also retarded in comparison to a normal regenerating lens of comparable age (Table 1, last column; and Table 3). In all other cases of tissue implants other than pituitary (whether the implant was in an eye contralateral to a pituitary implant eye or not), no secondary lens formation was present (Table 3; see Fig. 101.For example, a stage IX secondary lens has formed in an eye (Fig. 11) with a pituitary implant while in the contralateral eye (Fig. 12) containing a liver implant, the iris shows no indication of transformation. Seven control eyes were left unoperated or sham-operated (cornea1 slit made). No secondary lens formation was present in any of these eyes (contralateral to those receiving pituitary and exhibiting secondary lens formation). These results, in con-

VOLUME 52, 1976

junction with those mentioned above, indicate that the effect of the pituitary in the anterior chamber of the eye is due to a direct local action, as opposed to a systemic one. Lentectomies were performed on some of the eyes contralateral to those receiving pituitary implants. The stages of regeneration obtained at specific times following lentectomy were comparable to stages previously obtained in this laboratory and to those published by Yamada (196713)for the same species. Lens Polarity

Reversal

In all cases of secondary lens formation from the dorsal iris stimulated by pituitary implants, the polarity of the secondary lens was reversed. This analysis is based upon the criteria that normal polarity of the lens is reflected in: (11 primary depigmentation of the inner lamina of the iris first, (2) derivation of the cuboidal epithelium of the lens from the outer lamina of the iris, and the orientation of this epithelium toward the cornea1 side of the lens, and (3) derivation of the primary and secondary lens fiber forming cells from the inner lamina of the iris and orientation of these cells on the retinal side of the lens. Therefore the area of primary transformation of cells into lens fibers or the primary

FIG. 4. Section through an eye in which a pituitary (P) had been implanted 16 days prior to fixation. The pituitary is in intimate contact with the secondary lehs (SL), stage VI. The primary lens (PL) is intact and shows no evidence of deterioration as noted by lack of vacuolation and by the shattering of the central portion of the lens. The origin of the secondary lens, via the lens vesicle stalk, is the outer lamina of the dorsal iris, slightly dorsal to the pupillary margin. The active fiber pole of the secondary lens is shifted from the normal direction and oriented toward the pituitary implant. x 135. FIG. 5. Enlargement of Fig. 4, showing secondary lens in contact with the pituitary implant. The edge of the pupillary margin is visible at the bottom of the figure. Polarity is reversed in that the primary fibrogenic center is directed toward the pituitary implant. The arrow (-1 indicates fiber axis polarity. x 550. FIG. 6. Section through an eye, illustrating the category in Table 1, “wedged in cornea.” Pituitary (P) was implanted 16 days prior to sacrifice and can be seen wedged in the cornea. Stage III regenerate was growing from the dorsal iris. The primary lens (PL) is intact. Note the outer lamina of the dorsal iris is depigmented. x 135. FIG. 7. Section through an eye which illustrates the category in Table 1, “wedged in cornea.” Pituitary (Pl was implanted 9 days prior to sacrifice. The extent to which the implant is wedged in the cornea is less than that in the case illustrated in Fig. 6, and this difference (greater exposure of pituitary to anterior chamber fluid) is reflected in the extent of secondary lens formation (stage IV). The dorsal iris is swollen to form a vesicle.

Secondary

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DEVELOPMENTAL BIOLOGY

fibrogenic center is directed toward the retina. Reversed polarity was noted in all early stages of secondary lens formation since the outer lamina was first to depigment (Figs. 6 and 7). In some cases of later stages of secondary lens formation, the entire lens vesicle seems to have been derived from the outer lamina of the iris alone and the inner lamina and pupillary margin remained pigmented (Figs. 4, 5, and 11). Finally, polarity reversal is shown by the position of the primary and secondary lens fibers in later stages. The lens fibers and lens fiber forming cells, the primary fibrogenic center, is found on the pituitary (anterior chamber) side of the vesicle rather than the retinal side (Figs. 2-5, and 11) and often the cuboidal epithehum can be clearly seen on the retinal side of the vesicle (Fig. 5). Thus, the fiber axis is often positioned in the direction of the pituitary implant (Figs. 2 and 41. In one case in which the pituitary implant was found engulfing the ventral iris, both the inner and outer lamina of this portion of the iris margin showed depigmentation and swelling. DISCUSSION

The results of these experiments strongly suggest that there is a substance(s) that emanates from the implanted pituitary, causing iris transformation and secondary lens formation. Varia-

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bility in the results rests only in the extent to which regeneration has occurred and this can be correlated with the extent to which the dorsal iris was exposed to the pituitary implant tissue. These results thus extend the casual observation by Zalokar (1944) on the larval eye response to the presence of pituitary in culture and to those of Connely et al. (19731on explanted adult iris response to pituitary explants in vitro. The secondary lens formation, stimulated by adult pituitary implants, in these present experiments far exceeds the developmental stages observed in response to spinal ganglia implanted into the adult eye, where only depigmentation and swelling were observed in the iris tissue (Powell and Powers, 1973) and is equivalent to the regenerative ability exhibited by iris tissue, implanted into limb blastema (Reyer et al., 1973). It also appears from the present results that the pituitary secretes something that is capable of acting on iris tissue in some way which produces the same effect as the retinal substance, which supports lens regeneration in the absence of an adult lens and which emanates from neural retina implants in the presence of an intact lens (Williams and Higginbotham, 19751. Moreover, it is evident that the ectopic pituitary acts directly on the cells of the iris rather than through some type of systemic endocrine interaction since the implant has no positive influence on contra-

FIG. 8. Section through an eye in which a pituitary implant (P) was encapsulated in the cornea (Cl. Note the basement lamella (BL) surrounding the implant. No secondary lens formation is associated with the dorsal iris (DD. Primary lens (PL). The animal was sacrificed 18 days after implantation. x 135. FIG. 9. Section through an eye in which liver (1,) had been implanted 16 days prior to fixation. This is the eye contralateral to the case containing a pituitary implant shown in Figs. 4 and 5. Secondary lens formation (stage III) in this eye is retarded in comparison to that shown in Figs. 4 and 5. Primary lens (PL). Outer lamina of dorsal iris (OL). x 135. FIG. 10. Section through an eye in which liver tissue (L) had been implanted 18 days prior to fixation. The liver implant has adhered to the cornea (Cl but is located well inside the anterior chamber. No regeneration is seen from the dorsal iris (DI). x 135. FIG. 11. Section through an eye showing a 20-day pituitary implant (P) and a stage-IX secondary lens (SL). The pituitary appears larger in other sections but this section is used to best illustrate the reversed polarity of the secondary lens. x 135. FIG. 12. Section through the eye contralateral to the one shown in Fig. 11. The liver implant is situated well within the anterior chamber and there is no secondary lens formation from the dorsal iris (DIl. x 135.

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VOLUME 52, 1976

lateral eyes. These results, however, do pigmentation occurs in these cases, the iris not rule out the notion that, in typical lens cells showed enhancement of ribosomal regeneration, the pituitary may exert its RNA synthesis and initiation of DNA syninfluence through endocrine interaction thesis. Dispersed iris cells, maintained in (Schotti and Murphy, 1953). This consist- prolonged tissue culture, do form lenslike ent and clear-cut effect of the pituitary is structures (Eguchi, Abe, and Watanabe, somewhat unexpected since Schotti and 1974). Murphy (1953) demonstrated that hypophThus, we could suppose that a number of ysectomy caused a slight retardation, but substancesincluding those from the retina basically no other change in the regenera- and pituitary, are capable of disturbing or tive ability of the newt iris. However, shifting the delicate metabolic balance of neural and pituitary tissue clearly have these iris cells. The extent of the regenerasome effect on stimulating mitosis in re- tive response would reflect the quantity of generating tissue (Thornton, 1968). Fur- the substance(s) in the vicinity of the iris. thermore the pituitary functions differOr we might also consider the “transformently ectopically than it does in situ (Ma- ing” substance to be a general metabolic sur, 1969). Excess quantities of both product, secreted or excreted by many tisgrowth hormone and a prolactinlike hor- sues in different amounts. Under either mone are secreted by the pituitary trans- supposition, it would be possible for other planted to the throat region of the newt. actively metabolizing tissues to occasion-, Since the pituitary in situ acts as a stim- ally call forth such a response as did one ulator-y, albeit accessory, factor in both liver implant in the present set of experilens regeneration and in limb regenera- ments. tion, as was thoroughly discussed in the The polarity of the secondary developing Introduction, one could view its secretions lens was always reversed in the case of as one of many additive stimuli, acting on pituitary implants. More specifically, the the transformation, dedifferentiation, and primary fibrogenic center (the area of priregeneration in these systems. Thus, mary transformation of cells and lens fiber when the secretions of the pituitary are formation) was directed toward the pituipresent in large quantities, as may be the tary implant. In addition, other tissue imcase in the pituitary implants, they are plants that induce secondary lens formasufficient to support lens formation in the tion, such as the liver in one case in the absence of other necessary factors or in the present experiment, and spinal ganglia presence of inhibitory factors such as the (Powell and Powers, 1973) cause reversal intact lens. Indeed, there may not be one of lens polarity if implanted in the anterior specific chemical that triggers a response chamber. From the figures and figure legin the reacting tissue. The iris cells are ends (especially Figs. 15, 16, 19, and 201 programmed to give a regenerative re- published by Williams and Higginbotham sponse as a result of various changes ef- (1975) it appears that implantation of both fecting their metabolic state. Thus, adult and larval neural retina into the trauma inflicted by explantation of iris tis- anterior chamber also results in secondary sue into the body wall or body cavity may lens development with reversed polarity. Evidently the iris itself does not possess result in occasional lentoid production (Stone, 1958b). In vitro, early molecular inherent polarity for lens formation. Stone events of regeneration are stimulated sim- (1954) showed that iris segments turned ply by the process of culturing iris in Miniinside out, i.e., rotated 180” on the dorsomum Essential Medium, supplemented ventral axis, all developed with the fiber with 8% fetal calf serum (Reese, 1973;Jau- pole facing toward the retina, strongly ker and Yamada, 1972). Although no de- suggesting that the retina plays an impor-

POWELL AND SEGIL

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tant role in determining the medio-lateral axis of the lens. Similar reversal of polarity can occur in later stages of regeneration of the newt lens (Reyer, 1974) as it does in chick development when the antero-posterior axis is reversed in the &day embryo (Coulombre and Coulombre ,1963). We suggest that the chemical stimulus from the inducing tissue (retina, pituitary, or spinal ganglia) turns on a metabolic program in the cells (whether they be of the internal or external lamina of the iris) nearest the inducing tissue, thus establishing the active pole of the lens fiber axis. Even if there are several substances capable of supporting iris transformation and lens formation, it would be satisfying to identify at least one. With this goal in mind we are at present analyzing the differential effect of anterior and posterior pituitary implants and the effects of growth hormone-agar implants on the process of secondary lens formation. REFERENCES CAMERON, M. L., and STEELE, J. E. (1959). Simplified aldehydefuchsin staining of neurosecretory cells. Stain Tech. 34, 265-266. CONNELLY, T. G., ORTIZ, J. R., and YAMADA, T. (1973). Influence of the pituitary on Wolf&m lens regeneration. Develop. Biol. 31, 301-315. COPELAND, D. E. (1943). Cytology of the pituitary gland in developing and adult Triturus viridestens. J. Morph. 72, 379-409. COULOMBRE, J. L., and COULOMBRE, A. J. (1963). Lens development: Fiber elongation and lens orientation. Science 142, 1489-1490. DUMONT, J. N., and YAMADA, T. (1972). Differentiation of iris epithelial cells. Develop. Biol. 29, 385401. DUMONT, J. N., YAMADA, T., and CONE, M. V. (1970). Alteration of nucleolar ultrastructure in iris epithelial cells during initiation of Wolfflan lens regeneration. J. Exp. Zool. 174, 187-204. ECUCHI, G. (1961). The inhibitory effect of the injured and displaced lens formation in Triturus larvae. zmbryologia 6, 13-35. EGUCHI, G. (1963). Electron microscopic studies on lens regeneration. I. Mechanism of depigmentation of the iris. Embryologia 8, 45-62. EGUCHI, G. (1964). Electron microscopic studies on lens regeneration. II. Formation and growth of

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lens vesicle and differentiation of lens fibers. Embryologia 8, 247-287. EGUCHI, G., ABE, S., and WATANABE, K. (1974). Differentiation of lens-like structures from newt iris epithelial cells in vitro. Proc. Nat. Acad. Sci. USA 71, 5052-5056. HALL, A. B., and SCHOTT~, 0. E. (1951). Effects of hypophysectomies upon the initiation of regenerative processes in the limb of Triturus viridescens. J. Exp. Zool. 118, 368-388. JAUKER, F., and YAMADA, T. (1972). Progressive alteration in the pattern of nucleic acid metabolism in the iris cultured in vitro. J. Exp. Zool. 183, 145-153. KARASAKI, S. (1964). An electron microscopic study of Wolfian lens regeneration in the adult newt. J. Ultrastruct. Res. 11, 246-273. MASUR, S. K. (1969). Fine structure of the autotransplanted pituitary in the red eft, Notophthalmus viridescens. J. Comp. Endocrinol. 12, 12-32. POWELL, J. A., and POWERS, C. (1973). Effect on lens regeneration of implantation of spinal ganglia into the eyes of the newt, Notophthalmus. J. Exp. Zool. 183, 95-114. REESE, D. H. (1973). In vitro initiation in the newt iris of some early molecular events of lens regeneration. Exp. Eye Res. 17, 435-444. REYER, R. W. (1954). Regeneration of the lens of the amphibian eye. Quart. Rev. Biol. 29, l-46. REYER, R. W. (1971). DNA synthesis and the incorporation of labeled iris cells into the lens during lens regeneration in adult newts. Develop. Biol. 24, 533-558. REYER, R. W. (1974). Differentiation of lens fibers from lens epithelium in Ambystoma maculatum larvae and the repolarization of reversed, regenerating lenses in adult Notophthalmus uiridestens. Amer. Zool. 14, 1302. REYER, R. W., WOOLFITT, R. A., and WITHERSTY, L. T. (1973). Stimulation of lens regeneration from newt dorsal iris when implanted into the blastema of the regenerating limb. Develop. Biol. 32, 258281. SATO, N. L., and INOU~, S. (1973). Effects of growth hormone and nutrient on limb regeneration in hypophysectomized adult newts. J. Morph. 140, 477-486. SCHOTT~, 0. E. (1926). SystBme nerveux et rkgknbration chex le Triton. Rev. Suisse Zool. 33, 1-211. SCHOTTE, 0. E., and HALL, A. B. (1952). Effect of hypophysectomy upon phases of regeneration in progress (Triturus viridescens). J. Erp. Zool. 121, 521-560. SCHOTT~, 0. E., and MURPHY, G. W. (1953). Regeneration of the lens in absence of the pituitary in the adult newt (Triturus viridescens). J. Morph. 93, 447-464. SINGER, M. (1960). Nervous mechanisms in the re-

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generation of body parts in vertebrates. In “Developing Cell Systems and Their Control” (D. Rudnick, ed.), pp. 115-133. Eighteenth Growth Symposium. Ronald Press, New York. SINGER, M. (1965). A theory of the trophic nervous control of amphibian limb regeneration, including a re-evaluation of quantitative nerve requirements. In “Regeneration in Animals and Related Problems” (V. Kiortsis and H.A.L. Trampusch, eds.), pp. 20-32. North-Holland, Amsterdam. STONE, L. S. (1954). Further experiments on lens regeneration in eyes of the adult newt, Triturus V. viridescens. Anat. Rec. 120, 599-623. STONE, L. S. (1957). Regeneration of iris and lens in hypophysectomized adult newts. J. Exp. 2001. 136, 17-33. STONE, L. S. (1958a). Lens regeneration in adult newt eyes related to retina pigment cells and the neural retina factor. J. Exp. 2001. 139, 69-84. STONE,L. S. (1958b). Inhibition of lens regeneration in newt eyes by isolating the dorsal iris from the neural retina. Anat. Rec. 131, 151-172. STONE,L. S., and STEINITZ, H. (1953a). The regeneration of lenses in eyes with intact and regenerating retina in adult Triturus V. viridescens., Amer. Zool. 124, 435-467. STONE, L. S., and STEINITZ, H. (1953b). Effect of hypophysectomy and thyroidectomy on lens and retina regeneration in the adult newt, Triturw V. viridescens. J. Exp. Zool. 124, 469-504. TAKANO, K., YAMANAKA, G., and MIKAMI, Y. (1958). Wolffian lens regeneration in the eye containing a full grown lens in Triturus pyrrhogaster. Mie. Med. J. 8, 385-403. TASSAVA, R. A. (1969). Hormonal nutritional requirements for limb regeneration and survival of adult newts. J. Exp. Zool. 170, 33-50.

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THORNTON, C. (1968). Amphibian limb regeneration. Adv. in Morph. 7, 205-249. WILKERSON, J. A. (1963). The role of growth hormone in regeneration of the forelimb of the hypophysectomized newt. J. Exp. Zool. 154,223-230. WILLIAMS, L. A. (1970). The effect of a normal lens on lens regeneration in Notophthalmus viridestens viridescens. Amer. zool. 10, 322-323. WILLIAMS, L. A., and HIGGINBOTHAM, L. T. (1975). The role of a normal lens in Wolffian lens regeneration. J. Exp. Zool. 191, 233-251. YAMADA, T. (1966). Control of tissue specificity: The pattern of cellular synthetic activities in tissue transformation. Amer. Zool. 6, 21-31. YAMADA, T. (1967a). Cellular synthetic activities in induction of tissue transformation. In “Cell Differentiation”, pp. 116-130, Ciba Foundation Symposium, Williams and Wilkins, Baltimore. YAMADA, T. (1967b). Cellular and subcellular events in Wolffan lens regeneration. Zn “Current Topics in Developmental Biology” (A.A. Moscona and A. Monroy, eds.), Vol. 2, pp. 247-283. Academic Press, New York. YAMADA, T., and DUMONT, J. N. (1972). Macrophage activity in Wolffian lens regeneration. J. Morph. 136, 367-384. YAMADA, T., REESE, D. H., and MCDEVITT, D. S. (1973). Transformation of iris into lens in vitro and its dependency on neural retina. Difirentiation 1, 65-82. YAMADA, T., and MCDEVITT, D. S. (1974). Direct evidence for transformation of differentiated iris epithelial cells into lens cells. Develop. Biol. 38, 104-118. ZALOKAR, M. (1944). Contribution a l’etude de la regeneration du cristallin chez le Triton. Rev. Suisse Zool. 51, 443-521.