Exp. Eye Res. (1977) 24, 501-509
Repolarization
of Reversed, Regenerating Lenses in Adult Newts, Notophthalmus vividescens TV. REYER
RANDALL
Department of Anatomy, Sclzool of Medicilze, West Virginia Morgantown, W. Va. 26506, U.S.A.
University,
(Received9 September1976, Wew Yorlc) Following removal of the lens from eyes of adult newts, Notophthdmus viridescens, lens regeneration from the dorsal iris was allowed to proceed for 20-30 days. Then a circular disc of iris and the attached regenerating lens was excised, turned inside out, and replaced in the eye so that the lens fiber pole faced the cornea. Eyes were fixed from 1 to 30 days after the second operation. The lens epithelium, facing the vitreous body and neural retina, differentiated into new lens fibers which were laid down on the surface of the fibers present at the time of rotation. The remainder of the lens epithelium grew over the old lens fiber pole and covered the surface facing the cornea. In this way, normal polarity was re-established in the reversed, regeneratinglens. Key words: lens, regeneration, polarity, iris, eye, retina.
1. Introduction There is now considerableevidence that the normal polarity of the lensdependson the conditions present at its location in the pupil of the eye, namely vitreous and neural retina facing its posterior surface and aqueous humor bathing its anterior surface. Disturbances in these relationships initiate regulatory changes directed toward the restoration of normal polarity. Rotation of the lens in the B-day old chick embryo so that its antero-posterior axis was reversed resulted in differentiation of lens fibers from the lens epithelium facing the neural retina and growth of the lens epithelium from the lens equator over the old lens fibers which were now facing the cornea. The normal polarity of the lens was thereby restored (Coulombre and Coulombre, 1963; Coulombre, 1969). When the host lens was replaced by two donor lenses,each rotated 90” on its naso-temporal axis so that the anterior poles with lens epithelium were (1) facing each other, (2) facing away from each other or (3) facing in the same direction, then lens fibers differentiated from that part of the lens epithelium confronting the neural retina while the lens epithelium directed away from the neural retina remained, and its cells continued to divide. This epithelium subsequently spread over the lens fibers asfar as the eyecup margin. Nine days after the operation, t’he two lenses together had formed an ovoid body approaching a single, normal lens in shape and volume. Regardless of the original orientations, their polarities had regulated so that lens epithelium faced the cornea, lens fibers were in contact with the vitreous body and new equatorial zones lay adjacent to the margin of the eyecup (Coulombre and Coulombre, 1969). G&is-Galvez and Castro (1971) alsorotated lenses of 24-4-day-old chick embryos 180” to reverse the antero-posterior axis and then analyzed homogenates of the young lens fibers, which had developed from the lens epithelium after 4 days, by immunoelectrophoresisagainst antisera to total chick lens. There was an increase in the content of delta crystallin in these newly differentiated lens fibers. When a lens regenerates from the dorsal iris of a newt following extirpation of the 501
.x2
R.
W.
REYER
old lens, primary lens fibers differentiate from that part of the regenerating lens vesicle facing the neural retina and the lens epithelium forms from the remainder of the lens vesicle facing the cornea. Secondary lens fibers develop from the zone where lens epithelium meets lens fibers. This forms a new equatorial region adjacent to the pupillary margin of the iris (Reyer, 1954, 1962; Yamada, 1967). Control of this polarity depends on factors in the eyecup extrinsic to the iris. Stone (1954) reversed a segment of mid-dorsal iris by excising a square of iris, rotating it 180” on its dorsoventral axis and then replacing it, inside out, in the wound. After the iris had been allowed to heal for 8-13 days, the lens was removed. A new lens then regenerated from the rotated iris. The eyes were fixed 19 days after lentectomy. Seven out of 20 reversed irises regenerated a len s, among which three lenses with normal polarity were described. In these lenses, the lens epithelium faced the cornea, the lens fibers lay next to the vitreous, and secondary lens fibers were being added at a normal equatorial zone. Either single or double primary lens fiber centers were observed. In these experiments of Stone, the dorsal iris was in the reversed orientation throughout the entire course of lens regeneration. Since the Coulombres had observed repolarization following a similar reversal of a partly developed chick lens, it was important to ascertain whether a regenerating lens in which lens fibers were differentiating could also regulate its polarity when rotated inside out. Some of the results of these experiments have already been published in abstract form (Reyer, 1974). 2. Materials and Methods 911experiments were performed on adult newts, Noto1)htlbalnau.s vividescens, collected in West Virginia or Massachusetts and kept in the laboratory several months before use. After anaesthesia in (Fly/, or 0.2% chloretone, the animals were placed cm their sides in a deep Petri dish containing Holtfreter’s solution buffered with Tris-HCl to pH 7.0-7.3. In the first operation (Fig. l), the cornea was cut along the ventral margin with iridectomy scissors, the cornea1 flap reflected dorsally and the lens removed from the pupil with
Operation Incision cornea
I
Operation
in Lens
removed
2
Disc of iris with regenerating lens rototed inside-out and replaced in eye.
FIG. 1. Diagram of the operations on adult A70to$tkalmus vi?%lescena. In opsration 1, the oorneai incision is indicated by a dashed line. In operation 2, t,he re-opened cornea1 incision is indicated by the outer dashed line and the circular cut in the iris by the inner line of short dashes and dots. The regenerating lens is shown with a light stipple and the inner surface of the iris, rotated to face outwards, by a dark stipple.
REVERSED
REGEXERATING
503
LEXSES
watchmaker’s forceps. The cornea1 wound was closed, the animals were allowed to recover overnight on moist gauze at 12-15°C after which they were transferred to conditioned tap water. Lens regeneration proceeded for 20, 25 or 30 days. A second operation was then performed with the anaesthetized animal immersed in Amphibian Ringer’s solution using procedures similar to those in the first operation. The cornea1 scar was cut open and the cut extended nasally and temporally one-half to two-thirds the circumference of the cornea. A circular incision was made in the iris half way between the pupil and ora serrata. -4 disc of iris enclosing the small pupil and regenerating lens was removed, turned inside out and then replaced in the eye with the naso-temporal axis reversed (Fig. 1). The cornea1 flap was then replaced and its cut edges apposed so that the reversed iris was held in position. The animal was allowed to recover as before. TABLE
Rotation
I
of iris and regenerating lens inside Notophthalmus viridescent
out in adult
No. of eyes Lcntectomy to rotation
Series
1
Series 2
(days)
Rotation fixation (days)
to
20
“.-SO
s
8
2.5 30
5-30 3-30
9 8
7 7
25
I-17
33
Lens rotation
Itotded
lens
ECOVCIWl
31
In the fkt series of experiments, the iris and regenerating lens were reversed 20, 2.5 and 3~ days after lentectomy and the eyes were hxed in Bouin’s fluid 2-5 days after the second operation and then at 5-day intervals up to 30 days. In the second series, involving only 25-day lens regenerates, ca.ses were fixed at I- or 2-day intervals up to 17 days after operation two (Table I). Both eyes were lentectomized at operation one. As controls, some eyes were removed and fixed 20, 25 or 30 days after lentectomy at the same time as the iris was reversed in the contralateral eye. In other cases, the iris was reversed in only one eye at operation two, the lens being allowed to regenerate normally in the contralateral eye. Both eyes were then fixed in Bouin’s fluid at the same time. Serial sections (10 pm in thickness) were prepared from each paraffin embedded eye and stained with hematoxylin and erythrosin.
3. Results Series 1 The first series oE experiments was designed to determine the most suitable stage at which to reverse the regenerating lens. It was easentia.1 that the anterior lens epithelium and posterior lens fiber pole be aheady differentiated. At the same time, the regenerate should still be fixed in position by its attachment to the dorsal iris. Stages of regeneration best suited for this experiment would therefore be 8 to 10. As shown in Table II, the stages of lens regeneration ranged from 7 to 11 in control eyes fixed 20-30 days after lentectomy. Because stages 9 and 10 were observed after 25 days, this was the time interval between lentectomy and reversal of the lens and disc of iris selected for series 2.
504
When lens had lens fiber now had
R. TV. REYER
the eyes were fixed 2 or 3 days after lens and iris reversal, the regenerating retained its old polarity so that the lens epithelium faced the vitreous and the pole faced the cornea. In cases fixed 15 to 30 days after iris reversal, the lens normal polarity and had continued to differentiate and grow in size. A few TABLE
IX
Series 1: Stage of lens regeneration in the contralateral, lentectonaized, control eyes on day when th.e iris and regenerating lens was reversed in the experimental eyes
Days
of regeneration
20 25 30
7
4 -
8
-
* The stage designations refer to the morphological described by Sato (1940) and subsequently illustrated
Lens regeneration stage* 9 10 10g
1 2
3 1
11
--
3
stage series for lens regeneration by Reyor (1954, 1962) and Yamada
2
in the newt (1967).
lensesdegenerated. There w-eretoo few examples fixed after 5-15 days to follow the events during restitution of a normal polarity. A more detailed study of these changes on a larger number of eyes was ma’dein series2. Series 2 In all cases,the iris was reversed 25 days after lentectomy. In those control eyes which were fixed at this time, there were one stage 7, two stage 9, and six stage 10 lens regenerates (Fig. 2). One day after the disc of iris with the regenerating lens at its center had been removed and reimplanted inside out, the regenerating lens was still. attached to the dorsal, pupillary margin and lay in the pupil with the simple cuboidal lens epithelium facing the vitreous body and the lens fibers of the lens fiber pole adjacent to the cornea (Figs 3,4). The first indication of lens fiber differentiation from the lens epithelium consisted in elongation of these cells from cuboidal to columnar (Fig. 5) (two cases).Next, the elongating cells became more slender and came to lie with their long axes in apposition to the old lens fibers (Fig. 6). Adjacent to these, some columnar cells still remained (three cases).A new circular, equatorial zone thus became established where the elongating, columnar cells gradually changed orientation and formed a new layer of young lens fibers applied to the surface of the old lens fibers (two cases).These changesoccurred in that part of the lens epithelium exposed to the vitreous and neural retina. Meanwhile, there was a gradual growth of lens epithelium over the bare lens fibers adjacent to the cornea (Fig. 6). The timing of this latter event was quite variable. New lens fibers continued to differentiate from the lens epithelium (Fig. 7) gradually forming a sheath of lighter staining young fibers enclosing the posterior aspect,of the darker staining old lens fibers (Figs 8, 9). By this stage, the details of the initial events could no longer be ascertained. The surface of the Iens facing the cornea was now completely covered by a simple cuboidal lens epithelium so that the reversal of lens polarity was complete. For comparison, Fig. 10 illustrates a normal stage 104 lens regenerate. Growth of the lens in size continued
REVERSED
REGESERATING
LENSES
505
’ !, Fra. 2. A normal regenerating lens of stage 10: 2.5 days after extirpation of the original lens; left eye.. Cornea is t.o the right and neural retina to the left ( :< 130). Fro. 3. A stage 9. regenerating lens reversed so that the lens fiber pole faces the cornea, 1 clay after this operation, 26 days after lens removal: right eye ( x 60). Fra. 4. The same lens and iris as in Fig. 3 (x 130). FIG. 5. A stage 10, regenerating lens, 6 days after reversal and 31 days after lens removal; right eye. Cells of the lens epithclicm facing the vitreous are elongated (arrow). Cornea (c) (x 130).
506
R. W.
REYER
FIa. 6. A stage IO, regcneratin, ~7lem, 6 clays after 1‘t3 crsal anti 31 days sRcr lens remora.l; right eye. Beginning differentiation of lens fibers from lens epithelium (arrow). On the cornea1 side, to the left, the lens epitbelium is overgrowing the old lens fiber pole ( x 130). FIG. 7. A smaller, stage 10, regenerating lens, 8 days after reversal and 33 days after lens removal; right eye. New lens fibers (nf) are developing from the lens epithelium facing the vitreous (arrow). Corcea is to the left. Stroma of reversed iris (rst) ( x 130). FIG. 8. A stage 10, regenerating lens, 10 days after reversal and 35 days after lens removal; right eye. New lens fibers have differentiated from the lens epithelium (arrows). Lens fibers facing the cornea (6) are almost covered by lens epithelium ( x 130). FIG. 9. A stage log, regenerating lens, 12 days after reversal and 37 days after lens removal: left eye. New lens fibers which have developed from the lens epit,helium form a layer over the surface of the old lens fibers and also a concentric group adjacent to the iris (arrows). Cornea is to the right, vitreous to the left. (x 130).
REVERSED
REGENERATING
LEKSES
507
13 FIG. IO. A IIVL.I~~~~ stage IO;-: regclleratillg lens, 34 days after leos re:lloval: right eye. Cornea 1s to t,hu left, vitreous to the right. Kate uniform staining of lens fibers ( x 130). BIG. 11. A stage 11, regenerating lens, 15 days after reversal and 40 days after lens removal; right eye. Normal polarity has been restored. Cornea is to the left. Reversed iris has not healed to the remainder of the iris ( x 67). FIG. 12. The same reversed; regenerating lens as in Fig. Il. Sate the numerous nuclei throughout the layers of secondary lens fibers ( x 130). FIG. 13. A normal stage 11, regenerating lens, 40 days after lens rcmo~l in the left eye of the same animal as shown in Figs 11 and 12. Note the restriction of nuclei in the secondary lens fibers to the dorsal and rentral equatorial region (x 130).
508
R. W.
REYER
normally by the addition of newly different,iated lens fibers at the equatorial zone. There was a loss of the discontinuity in staining intensity between the lens fibers laid down before and after rotation. The only feature distinguishing the reversed lens from a normal one was a greater abundance of nuclei among the lens fibers at the anterior pole in the former (Figs 11, 12, 13).
4. Discussion The pupil of the lentectomized eye appears to be a region in which polarized environmental stimuli have a strong morphogenetic effect on a developing lens. Abundant experimental evidence (Stone, 1965; Reyer, 1954, 1962, 1977a; Scheib, 1965) points to the neural retina as the source of this stimulus. The chemical nature of the st.imulus is unknown but it must traverse the vitreous body to act on t,he iris t.issue which comes into contact with the anterior surface of this gel-like material following removal of the lens. Under this influence, RNA and protein synthesis in the dorsal iris epithelium is augmented and these cells re-enter the cell-cycle and dedifferent,iate. Initially, a lens vesicle, composed of a uniform population of depigmented cells, grows from the pupillary margin of the iris into the pupillary space. The subsequent differentiation of nondividing lens fibers from the posterior cells of this vesicle and of rapidly dividing lens epithelial cells from the anterior cells apparently is controlled by the action of the neural retina1 st.imulus on one side of the vesicle only, that is, the side facing the vitreous body. The further growth of the lens and the laying down of secondary lens fibers in t,he equatorial region next to the pupillary margin of the iris occur under this same polarized stimulus and result in a regenerat,ed lens of normal morphology and orientation which occupies the pupil and re-establishes a. normal refractive path for formation of the visual image. Manipulations which alter these relationships result in changes in lens polarity, Thus, implants of neural retina (Williams and Higginbotham, 1975) or pituitary tissue (Powell and Segil; 1976) i.nto the anterior chamber of adult newt eyes with intact lens sometimes stimulated early stages of lens regeneration from the dorsal iris. If lens fiber formation was initiated, the lens fiber pole was always directed toward the source of the stimulus (neural retina or pituita,ry). Xany years ago, Mikami (1941) observed t,hat iris implants into lentectomized eyes regenerated lenses most, frequently when located in the pupil. Implants lying deep within the vitreous body were deficient in lens epithelium while there was minimal lens fiber differentiation from implants lying in the anterior chamber. This polarized stimulus in the pupil also tends to bring a!bout the regulation of abnormally oriented tissues toward a normal lens morphology as following the inside-out reversal of dorsal iris by Stone (1954), or of the regenerating lens in the results reported here as well as implanted lens epithelium randomly oriented in the pupillary space (Reyer, 1977b). It can be concluded that the one-sided source of the stimulus for lens regeneration in the pupil is important in controlling the reconstitution of a normal regenerate and therefore in the re-establishment of visual acuity. ACKNOWLEDGMENTS
The author wishes to acknowledge the technical assistance of Mrs Alice Barnett and Mrs Dorothy Heritage. This investigation was supported by Research Grant EY 00196 from the National Eye Institute, National Institutes of Health.
REVERSED
REGESERATING
LENSES
509
REFERENCES Coulombre, A. J. (1969). Regulation of ocular morphogenesis. Inzqest. OphthaZmoZ. 8, 25-31. Couiombre, J. L. and Coulombre, A. J. (1963). Lens development. I. Fiber elongation and lens orientation. Science 142, 1489-90. Coulombre, J. L. and Coulombre, A. J. (1969). Lens development. IV. Size, shape, and orientation. Invest. Ophthalmol. 8, 251-7. G&is-Galvez, J. X. and Castro, J. M. (1971). Protein biosynthesis after lens rotation: an immunoelectrophoretic analysis in the chick embryo. J. Ez~. Zool. 177, 313-18. Mikami, Y. (1941). Experimental analysis of the Wolffian lens-regeneration in adult newt, Triturus pyrrhogaster. Jap. J. Zool. 9, 269-302. Powell. J. A. and Segil, N. (1976). Secondary lens formation caused by implantation of pituitary into the eyes of the newt, Notophtha~lmus. Develop. Biol. 52, 128-40. Reyer, R’. W. (1954). Regeneration of the lens in the amphibian eye. Quart. Xev. Biol. 29, l-&6. Reyer, R. W. (1962). Regeneration in the amphibian eye. In Regeneration (20th Growth Symposium). (Ed. R&nick, D.). Pp. 211-65. Ronald Press, New York. Reyer, R. W. (1974). Differentiation of lens fibers from lens epithelium in Bmby&orna mnculntum larvae and the repolarization of reversed, regenerating lenses in adult A~otophthalmus viridescens. Am. Zool. 14, 1302. Reyer, R. W. (1977a). The amphibian eye, development and regeneration. In %ndbooL of Xen.sory Physiology VII/S, The Visual Xystem in Eelolution, Part A. Vertebrates (Ed. Crescitelli, F.). Springer-Verlag, Berlin. (In press). Reyer, R. W. (1977b). Morphological evidence for lens differentiation from intraocular implants of Iens epithelium in dmbystoma. naac~~latum,. Ezp. Eye Res. 24, 511-22. Sato, T. (1940). VergIeichende Studien sber die Gesehwindigkeit der Wolf&hen Linsenregeneration bei Triton trreniatus und bei Diemyctylus pyrrhogaster. Wilhelm Rous’ Arch. htwicld-Nech, Org. 140, 570-613. Scheib, D. (1965). Recherches recentes sur la regeneration du cristallin chez les vertebr&. Evolution du probleme entre 1931 et 1963. Ergebn. Anat. Entwickl.-Gesch. 38, 46-114. Stone, L. S. (1954). Further experiments on lens regeneration in eyes of the adult newt Triturus 1,. viridescens. Anat. Rec. 120, 599-624. Stone, L. S. (1965). The regeneration of the crystalline lens. Inoest. Ophdhal~mol. 4, 420-32. Williams, L. A. and Higginbotham, L. T. (1975). The role of a normal lens in Wolffian lens regeneration. J. Exp. Zool. 191, 233-52. Ya,mada, T. (1967). Cellular and subcellular events in Wolffian lens regeneration. Cum. Top. Develop. Biol. 2, 245-83.
Repolarization
of Reversed, Regenerating Lenses in Adult Newts, Notophthalmus vividescens TV. REYER
RANDALL
Department of Anatomy, Sclzool of Medicilze, West Virginia Morgantown, W. Va. 26506, U.S.A.
University,
(Received9 September1976, Wew Yorlc) Following removal of the lens from eyes of adult newts, Notophthdmus viridescens, lens regeneration from the dorsal iris was allowed to proceed for 20-30 days. Then a circular disc of iris and the attached regenerating lens was excised, turned inside out, and replaced in the eye so that the lens fiber pole faced the cornea. Eyes were fixed from 1 to 30 days after the second operation. The lens epithelium, facing the vitreous body and neural retina, differentiated into new lens fibers which were laid down on the surface of the fibers present at the time of rotation. The remainder of the lens epithelium grew over the old lens fiber pole and covered the surface facing the cornea. In this way, normal polarity was re-established in the reversed, regeneratinglens. Key words: lens, regeneration, polarity, iris, eye, retina.
1. Introduction There is now considerableevidence that the normal polarity of the lensdependson the conditions present at its location in the pupil of the eye, namely vitreous and neural retina facing its posterior surface and aqueous humor bathing its anterior surface. Disturbances in these relationships initiate regulatory changes directed toward the restoration of normal polarity. Rotation of the lens in the B-day old chick embryo so that its antero-posterior axis was reversed resulted in differentiation of lens fibers from the lens epithelium facing the neural retina and growth of the lens epithelium from the lens equator over the old lens fibers which were now facing the cornea. The normal polarity of the lens was thereby restored (Coulombre and Coulombre, 1963; Coulombre, 1969). When the host lens was replaced by two donor lenses,each rotated 90” on its naso-temporal axis so that the anterior poles with lens epithelium were (1) facing each other, (2) facing away from each other or (3) facing in the same direction, then lens fibers differentiated from that part of the lens epithelium confronting the neural retina while the lens epithelium directed away from the neural retina remained, and its cells continued to divide. This epithelium subsequently spread over the lens fibers asfar as the eyecup margin. Nine days after the operation, t’he two lenses together had formed an ovoid body approaching a single, normal lens in shape and volume. Regardless of the original orientations, their polarities had regulated so that lens epithelium faced the cornea, lens fibers were in contact with the vitreous body and new equatorial zones lay adjacent to the margin of the eyecup (Coulombre and Coulombre, 1969). G&is-Galvez and Castro (1971) alsorotated lenses of 24-4-day-old chick embryos 180” to reverse the antero-posterior axis and then analyzed homogenates of the young lens fibers, which had developed from the lens epithelium after 4 days, by immunoelectrophoresisagainst antisera to total chick lens. There was an increase in the content of delta crystallin in these newly differentiated lens fibers. When a lens regenerates from the dorsal iris of a newt following extirpation of the 501
.x2
R.
W.
REYER
old lens, primary lens fibers differentiate from that part of the regenerating lens vesicle facing the neural retina and the lens epithelium forms from the remainder of the lens vesicle facing the cornea. Secondary lens fibers develop from the zone where lens epithelium meets lens fibers. This forms a new equatorial region adjacent to the pupillary margin of the iris (Reyer, 1954, 1962; Yamada, 1967). Control of this polarity depends on factors in the eyecup extrinsic to the iris. Stone (1954) reversed a segment of mid-dorsal iris by excising a square of iris, rotating it 180” on its dorsoventral axis and then replacing it, inside out, in the wound. After the iris had been allowed to heal for 8-13 days, the lens was removed. A new lens then regenerated from the rotated iris. The eyes were fixed 19 days after lentectomy. Seven out of 20 reversed irises regenerated a len s, among which three lenses with normal polarity were described. In these lenses, the lens epithelium faced the cornea, the lens fibers lay next to the vitreous, and secondary lens fibers were being added at a normal equatorial zone. Either single or double primary lens fiber centers were observed. In these experiments of Stone, the dorsal iris was in the reversed orientation throughout the entire course of lens regeneration. Since the Coulombres had observed repolarization following a similar reversal of a partly developed chick lens, it was important to ascertain whether a regenerating lens in which lens fibers were differentiating could also regulate its polarity when rotated inside out. Some of the results of these experiments have already been published in abstract form (Reyer, 1974). 2. Materials and Methods 911experiments were performed on adult newts, Noto1)htlbalnau.s vividescens, collected in West Virginia or Massachusetts and kept in the laboratory several months before use. After anaesthesia in (Fly/, or 0.2% chloretone, the animals were placed cm their sides in a deep Petri dish containing Holtfreter’s solution buffered with Tris-HCl to pH 7.0-7.3. In the first operation (Fig. l), the cornea was cut along the ventral margin with iridectomy scissors, the cornea1 flap reflected dorsally and the lens removed from the pupil with
Operation Incision cornea
I
Operation
in Lens
removed
2
Disc of iris with regenerating lens rototed inside-out and replaced in eye.
FIG. 1. Diagram of the operations on adult A70to$tkalmus vi?%lescena. In opsration 1, the oorneai incision is indicated by a dashed line. In operation 2, t,he re-opened cornea1 incision is indicated by the outer dashed line and the circular cut in the iris by the inner line of short dashes and dots. The regenerating lens is shown with a light stipple and the inner surface of the iris, rotated to face outwards, by a dark stipple.
REVERSED
REGEXERATING
503
LEXSES
watchmaker’s forceps. The cornea1 wound was closed, the animals were allowed to recover overnight on moist gauze at 12-15°C after which they were transferred to conditioned tap water. Lens regeneration proceeded for 20, 25 or 30 days. A second operation was then performed with the anaesthetized animal immersed in Amphibian Ringer’s solution using procedures similar to those in the first operation. The cornea1 scar was cut open and the cut extended nasally and temporally one-half to two-thirds the circumference of the cornea. A circular incision was made in the iris half way between the pupil and ora serrata. -4 disc of iris enclosing the small pupil and regenerating lens was removed, turned inside out and then replaced in the eye with the naso-temporal axis reversed (Fig. 1). The cornea1 flap was then replaced and its cut edges apposed so that the reversed iris was held in position. The animal was allowed to recover as before. TABLE
Rotation
I
of iris and regenerating lens inside Notophthalmus viridescent
out in adult
No. of eyes Lcntectomy to rotation
Series
1
Series 2
(days)
Rotation fixation (days)
to
20
“.-SO
s
8
2.5 30
5-30 3-30
9 8
7 7
25
I-17
33
Lens rotation
Itotded
lens
ECOVCIWl
31
In the fkt series of experiments, the iris and regenerating lens were reversed 20, 2.5 and 3~ days after lentectomy and the eyes were hxed in Bouin’s fluid 2-5 days after the second operation and then at 5-day intervals up to 30 days. In the second series, involving only 25-day lens regenerates, ca.ses were fixed at I- or 2-day intervals up to 17 days after operation two (Table I). Both eyes were lentectomized at operation one. As controls, some eyes were removed and fixed 20, 25 or 30 days after lentectomy at the same time as the iris was reversed in the contralateral eye. In other cases, the iris was reversed in only one eye at operation two, the lens being allowed to regenerate normally in the contralateral eye. Both eyes were then fixed in Bouin’s fluid at the same time. Serial sections (10 pm in thickness) were prepared from each paraffin embedded eye and stained with hematoxylin and erythrosin.
3. Results Series 1 The first series oE experiments was designed to determine the most suitable stage at which to reverse the regenerating lens. It was easentia.1 that the anterior lens epithelium and posterior lens fiber pole be aheady differentiated. At the same time, the regenerate should still be fixed in position by its attachment to the dorsal iris. Stages of regeneration best suited for this experiment would therefore be 8 to 10. As shown in Table II, the stages of lens regeneration ranged from 7 to 11 in control eyes fixed 20-30 days after lentectomy. Because stages 9 and 10 were observed after 25 days, this was the time interval between lentectomy and reversal of the lens and disc of iris selected for series 2.
504
When lens had lens fiber now had
R. TV. REYER
the eyes were fixed 2 or 3 days after lens and iris reversal, the regenerating retained its old polarity so that the lens epithelium faced the vitreous and the pole faced the cornea. In cases fixed 15 to 30 days after iris reversal, the lens normal polarity and had continued to differentiate and grow in size. A few TABLE
IX
Series 1: Stage of lens regeneration in the contralateral, lentectonaized, control eyes on day when th.e iris and regenerating lens was reversed in the experimental eyes
Days
of regeneration
20 25 30
7
4 -
8
-
* The stage designations refer to the morphological described by Sato (1940) and subsequently illustrated
Lens regeneration stage* 9 10 10g
1 2
3 1
11
--
3
stage series for lens regeneration by Reyor (1954, 1962) and Yamada
2
in the newt (1967).
lensesdegenerated. There w-eretoo few examples fixed after 5-15 days to follow the events during restitution of a normal polarity. A more detailed study of these changes on a larger number of eyes was ma’dein series2. Series 2 In all cases,the iris was reversed 25 days after lentectomy. In those control eyes which were fixed at this time, there were one stage 7, two stage 9, and six stage 10 lens regenerates (Fig. 2). One day after the disc of iris with the regenerating lens at its center had been removed and reimplanted inside out, the regenerating lens was still. attached to the dorsal, pupillary margin and lay in the pupil with the simple cuboidal lens epithelium facing the vitreous body and the lens fibers of the lens fiber pole adjacent to the cornea (Figs 3,4). The first indication of lens fiber differentiation from the lens epithelium consisted in elongation of these cells from cuboidal to columnar (Fig. 5) (two cases).Next, the elongating cells became more slender and came to lie with their long axes in apposition to the old lens fibers (Fig. 6). Adjacent to these, some columnar cells still remained (three cases).A new circular, equatorial zone thus became established where the elongating, columnar cells gradually changed orientation and formed a new layer of young lens fibers applied to the surface of the old lens fibers (two cases).These changesoccurred in that part of the lens epithelium exposed to the vitreous and neural retina. Meanwhile, there was a gradual growth of lens epithelium over the bare lens fibers adjacent to the cornea (Fig. 6). The timing of this latter event was quite variable. New lens fibers continued to differentiate from the lens epithelium (Fig. 7) gradually forming a sheath of lighter staining young fibers enclosing the posterior aspect,of the darker staining old lens fibers (Figs 8, 9). By this stage, the details of the initial events could no longer be ascertained. The surface of the Iens facing the cornea was now completely covered by a simple cuboidal lens epithelium so that the reversal of lens polarity was complete. For comparison, Fig. 10 illustrates a normal stage 104 lens regenerate. Growth of the lens in size continued
REVERSED
REGESERATING
LENSES
505
’ !, Fra. 2. A normal regenerating lens of stage 10: 2.5 days after extirpation of the original lens; left eye.. Cornea is t.o the right and neural retina to the left ( :< 130). Fro. 3. A stage 9. regenerating lens reversed so that the lens fiber pole faces the cornea, 1 clay after this operation, 26 days after lens removal: right eye ( x 60). Fra. 4. The same lens and iris as in Fig. 3 (x 130). FIG. 5. A stage 10, regenerating lens, 6 days after reversal and 31 days after lens removal; right eye. Cells of the lens epithclicm facing the vitreous are elongated (arrow). Cornea (c) (x 130).
506
R. W.
REYER
FIa. 6. A stage IO, regcneratin, ~7lem, 6 clays after 1‘t3 crsal anti 31 days sRcr lens remora.l; right eye. Beginning differentiation of lens fibers from lens epithelium (arrow). On the cornea1 side, to the left, the lens epitbelium is overgrowing the old lens fiber pole ( x 130). FIG. 7. A smaller, stage 10, regenerating lens, 8 days after reversal and 33 days after lens removal; right eye. New lens fibers (nf) are developing from the lens epithelium facing the vitreous (arrow). Corcea is to the left. Stroma of reversed iris (rst) ( x 130). FIG. 8. A stage 10, regenerating lens, 10 days after reversal and 35 days after lens removal; right eye. New lens fibers have differentiated from the lens epithelium (arrows). Lens fibers facing the cornea (6) are almost covered by lens epithelium ( x 130). FIG. 9. A stage log, regenerating lens, 12 days after reversal and 37 days after lens removal: left eye. New lens fibers which have developed from the lens epit,helium form a layer over the surface of the old lens fibers and also a concentric group adjacent to the iris (arrows). Cornea is to the right, vitreous to the left. (x 130).
REVERSED
REGENERATING
LEKSES
507
13 FIG. IO. A IIVL.I~~~~ stage IO;-: regclleratillg lens, 34 days after leos re:lloval: right eye. Cornea 1s to t,hu left, vitreous to the right. Kate uniform staining of lens fibers ( x 130). BIG. 11. A stage 11, regenerating lens, 15 days after reversal and 40 days after lens removal; right eye. Normal polarity has been restored. Cornea is to the left. Reversed iris has not healed to the remainder of the iris ( x 67). FIG. 12. The same reversed; regenerating lens as in Fig. Il. Sate the numerous nuclei throughout the layers of secondary lens fibers ( x 130). FIG. 13. A normal stage 11, regenerating lens, 40 days after lens rcmo~l in the left eye of the same animal as shown in Figs 11 and 12. Note the restriction of nuclei in the secondary lens fibers to the dorsal and rentral equatorial region (x 130).
508
R. W.
REYER
normally by the addition of newly different,iated lens fibers at the equatorial zone. There was a loss of the discontinuity in staining intensity between the lens fibers laid down before and after rotation. The only feature distinguishing the reversed lens from a normal one was a greater abundance of nuclei among the lens fibers at the anterior pole in the former (Figs 11, 12, 13).
4. Discussion The pupil of the lentectomized eye appears to be a region in which polarized environmental stimuli have a strong morphogenetic effect on a developing lens. Abundant experimental evidence (Stone, 1965; Reyer, 1954, 1962, 1977a; Scheib, 1965) points to the neural retina as the source of this stimulus. The chemical nature of the st.imulus is unknown but it must traverse the vitreous body to act on t,he iris t.issue which comes into contact with the anterior surface of this gel-like material following removal of the lens. Under this influence, RNA and protein synthesis in the dorsal iris epithelium is augmented and these cells re-enter the cell-cycle and dedifferent,iate. Initially, a lens vesicle, composed of a uniform population of depigmented cells, grows from the pupillary margin of the iris into the pupillary space. The subsequent differentiation of nondividing lens fibers from the posterior cells of this vesicle and of rapidly dividing lens epithelial cells from the anterior cells apparently is controlled by the action of the neural retina1 st.imulus on one side of the vesicle only, that is, the side facing the vitreous body. The further growth of the lens and the laying down of secondary lens fibers in t,he equatorial region next to the pupillary margin of the iris occur under this same polarized stimulus and result in a regenerat,ed lens of normal morphology and orientation which occupies the pupil and re-establishes a. normal refractive path for formation of the visual image. Manipulations which alter these relationships result in changes in lens polarity, Thus, implants of neural retina (Williams and Higginbotham, 1975) or pituitary tissue (Powell and Segil; 1976) i.nto the anterior chamber of adult newt eyes with intact lens sometimes stimulated early stages of lens regeneration from the dorsal iris. If lens fiber formation was initiated, the lens fiber pole was always directed toward the source of the stimulus (neural retina or pituita,ry). Xany years ago, Mikami (1941) observed t,hat iris implants into lentectomized eyes regenerated lenses most, frequently when located in the pupil. Implants lying deep within the vitreous body were deficient in lens epithelium while there was minimal lens fiber differentiation from implants lying in the anterior chamber. This polarized stimulus in the pupil also tends to bring a!bout the regulation of abnormally oriented tissues toward a normal lens morphology as following the inside-out reversal of dorsal iris by Stone (1954), or of the regenerating lens in the results reported here as well as implanted lens epithelium randomly oriented in the pupillary space (Reyer, 1977b). It can be concluded that the one-sided source of the stimulus for lens regeneration in the pupil is important in controlling the reconstitution of a normal regenerate and therefore in the re-establishment of visual acuity. ACKNOWLEDGMENTS
The author wishes to acknowledge the technical assistance of Mrs Alice Barnett and Mrs Dorothy Heritage. This investigation was supported by Research Grant EY 00196 from the National Eye Institute, National Institutes of Health.
REVERSED
REGESERATING
LENSES
509
REFERENCES Coulombre, A. J. (1969). Regulation of ocular morphogenesis. Inzqest. OphthaZmoZ. 8, 25-31. Couiombre, J. L. and Coulombre, A. J. (1963). Lens development. I. Fiber elongation and lens orientation. Science 142, 1489-90. Coulombre, J. L. and Coulombre, A. J. (1969). Lens development. IV. Size, shape, and orientation. Invest. Ophthalmol. 8, 251-7. G&is-Galvez, J. X. and Castro, J. M. (1971). Protein biosynthesis after lens rotation: an immunoelectrophoretic analysis in the chick embryo. J. Ez~. Zool. 177, 313-18. Mikami, Y. (1941). Experimental analysis of the Wolffian lens-regeneration in adult newt, Triturus pyrrhogaster. Jap. J. Zool. 9, 269-302. Powell. J. A. and Segil, N. (1976). Secondary lens formation caused by implantation of pituitary into the eyes of the newt, Notophtha~lmus. Develop. Biol. 52, 128-40. Reyer, R’. W. (1954). Regeneration of the lens in the amphibian eye. Quart. Xev. Biol. 29, l-&6. Reyer, R. W. (1962). Regeneration in the amphibian eye. In Regeneration (20th Growth Symposium). (Ed. R&nick, D.). Pp. 211-65. Ronald Press, New York. Reyer, R. W. (1974). Differentiation of lens fibers from lens epithelium in Bmby&orna mnculntum larvae and the repolarization of reversed, regenerating lenses in adult A~otophthalmus viridescens. Am. Zool. 14, 1302. Reyer, R. W. (1977a). The amphibian eye, development and regeneration. In %ndbooL of Xen.sory Physiology VII/S, The Visual Xystem in Eelolution, Part A. Vertebrates (Ed. Crescitelli, F.). Springer-Verlag, Berlin. (In press). Reyer, R. W. (1977b). Morphological evidence for lens differentiation from intraocular implants of Iens epithelium in dmbystoma. naac~~latum,. Ezp. Eye Res. 24, 511-22. Sato, T. (1940). VergIeichende Studien sber die Gesehwindigkeit der Wolf&hen Linsenregeneration bei Triton trreniatus und bei Diemyctylus pyrrhogaster. Wilhelm Rous’ Arch. htwicld-Nech, Org. 140, 570-613. Scheib, D. (1965). Recherches recentes sur la regeneration du cristallin chez les vertebr&. Evolution du probleme entre 1931 et 1963. Ergebn. Anat. Entwickl.-Gesch. 38, 46-114. Stone, L. S. (1954). Further experiments on lens regeneration in eyes of the adult newt Triturus 1,. viridescens. Anat. Rec. 120, 599-624. Stone, L. S. (1965). The regeneration of the crystalline lens. Inoest. Ophdhal~mol. 4, 420-32. Williams, L. A. and Higginbotham, L. T. (1975). The role of a normal lens in Wolffian lens regeneration. J. Exp. Zool. 191, 233-52. Ya,mada, T. (1967). Cellular and subcellular events in Wolffian lens regeneration. Cum. Top. Develop. Biol. 2, 245-83.
