/. Embryo/, exp. Morph. Vol. 48, pp. 205-214, 1978
Printed in Great Britain © Company of Biologists Limited 1978
205
Lens regeneration from cornea of larval Xenopus
laevis in the presence of the lens
By JULIE G. REEVE AND ARTHUR E. WILD
From the Department of Biology, University of Southampton
SUMMARY
In Xenopus laevis tadpoles, wounding of the outer cornea failed to initiate lens regeneration.
If both the outer and inner corneas were wounded or if the lens was dislocated, lens
regeneration was initiated but failed to continue beyond stage III. However, lensectomy
followed by re-implantation of the lens resulted in the regeneration of a fully differentiated
lens in several cases, despite the presence of the re-implanted lens. Although some of the
regenerates in these eyes were also arrested at stage III, those which attained full lens
differentiation, i.e. stage V, developed normally and synthesized crystallins from the onset
of stage IV as indicated by a positive immunofluorescence reaction.
Histological examination of the dislocated and re-implanted lenses showed the majority
of them to be normal in appearance.
Cornea transplanted to the posterior chamber of the eye also regenerated a lens in the
presence of the re-implanted lens. All these regenerates underwent lens fibre differentiation
to give stage-V regenerates.
These findings show that lens regeneration from the cornea can occur in the presence
of lens. Results are discussed on the basis that contrary to earlier suggestions, an inhibitory
lens factor does not exist in vivo, but rather that a factor for the initiation and maintenance
of regeneration emanates from the eye cup and upon wounding of the inner cornea is able
to reach the inner cell layer of the outer cornea and initiate lens regeneration.
INTRODUCTION
In larval Xenopus laevis, lensectomy may be followed by regeneration of a
new lens from the inner cell layer of the outer cornea and both the eye cup
and the inner cornea are believed to be of importance in this process (Freeman,
1963). Thus lensectomy combined with eye cup removal failed to initiate lens
regeneration, and re-growth of the inner cornea between the eye cup and the
outer cornea at a stage before the epithelial lens vesicle had formed, prevented
further development of the vesicle.
There have been subsequently few studies attempting to evaluate the roles
of the various ocular tissues in the initiation and control of in vivo lens regeneration from the cornea. It has been shown that cornea transplanted alone to
certain ectopic sites (i.e. tail fin) fails to regenerate lens (Waggoner, 1973).
However, we have previously shown that cornea of ectopic eyes grown in the
1
Authors' address: Department of Biology, Medical and Biological Sciences Building,
University of Southampton, Bassett Crescent E., Southampton SO9 3TU, U.K.
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J. G. REEVE AND A. E. WILD
tail is able to undergo lens regeneration (Reeve & Wild, 1977) thus confirming
the importance of the eye environment.
In vitro studies have demonstrated the capacity of cultured cornea to regenerate
lens in the absence of other eye tissues (Campbell & Jones, 1968). Indeed, the
inclusion of retina or retinal extract in the culture system appeared to significantly inhibit lens regeneration as did inclusion of lens or lens extract
(Manwaring, 1972). This latter finding seems to be consistent with the existence
of an inhibitory lens factor which Stone (1965) and Stone & Vultee (1949)
postulated from studies on Wolffian lens regeneration. Campbell & Jones
(1968) and Manwaring (1972) have also suggested that this factor may play
a major role in the control of lens regeneration from the cornea in vivo.
Since there is discrepancy between results obtained in vitro and results
obtained in vivo regarding the importance of the eye cup/retina in the control
of lens regeneration, we feel that one cannot confidently extrapolate from one
system to the other. Therefore the present study attempts to re-examine the
relative roles of the lens and the eye cup in the control of lens regeneration
from the cornea in vivo.
MATERIALS AND METHODS
Tadpoles at stages 49-51 were anaesthetized in 1:3000 MS222 in 10 % Holtfreter's solution and operated on in sterile full-strength Holtfreter's solution
where they remained for 24 h. They were then subsequently reared in 10 %
Holtfreter's solution. Experiments were carried out in the following manner.
(1) Corneal wounding
(a) A semicircular incision was made with corneal suture scissors through
the outer cornea along the corneal-epidermal junction of the right eye, the
outer cornea reflected dorsally, and the attachment between the inner and
outer cornea severed. The inner cornea itself remained intact. (See Fig. 1 for
a diagrammatic representation of the relationship between inner and outer
cornea in the tadpole eye.)
(b) The outer cornea was again reflected dorsally as described above and
an incision also made in the inner cornea.
Tadpoles were sacrificed 10 or 20 days after wounding.
(2) Lens dislocation
The outer cornea was reflected dorsally and an incision made in the inner
cornea as described in (1). The lens was dislocated by applying pressure to
the back of the eye until at least half the diameter of the lens bulged through
the pupillary space. Tadpoles were sacrificed at 10 or 20 days after the operation.
Lens regeneration from, cornea of larval Xenopus
207
Fig. 1. Diagrammatic representation of a section through the eye of a stage-50
tadpole showing the outer (OC) and inner (IC) cornea and their point of attachment
(PA).
(3) Lensectomy followed by re-implantation of the lens
Incisions were made in the outer and inner corneas as described in (1).
Pressure was then applied with forceps to the back of the eye and the lens
forced through. The pupillary space was then slightly widened with forceps
and the extruded lens carefully re-inserted into the eye. Tadpoles were sacrificed
at 6, 10 or 20 days after the operation.
(4) Implantation of cornea into the posterior eye chamber
Lensectomy was performed in host animals as described under (3). Cornea
(together with some attached pericorneal epidermis) that had been removed
from stage-49-51 donor animals was implanted into the posterior eye chamber
of the lensectomized eye and the lens carefully re-inserted into the eye. Tadpoles
were sacrificed 14 days later.
Controls
Control lensectomies, involving permanent removal of the lens, were carried
out for the above experiments on stage-50 tadpoles.
14-2
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J. G. REEVE AND A. E. WILD
Histological procedures
With the exception of those used for immunofluorescence studies described
below, tadpoles were fixed in 10 % formol-saline and embedded in paraffin
wax. Tissues were sectioned serially at 7 /tm and stained with Erlich's
haematoxylin and eosin.
Detection of lens crystallin synthesis
Tadpoles subjected to the procedure described in (3) were anaesthetized,
decapitated and the heads washed in water at 4 °C. The heads were fixed in
cold 95 % ethanol and processed for immunofluorescence staining according
to the method described by Sainte-Marie (1962). A rabbit antiserum to adult
Xenopus whole-lens crystallins was prepared according to the method described
in Billett & Wild (1975) and the y-globulin fraction conjugated to F.I.T.C.
This conjugate was then absorbed with pig liver powder to reduce possible
non-specific staining reactions. Sections were observed using a Leitz fluorescence
microscope with a Ploem vertical epi-illumination attachment and photographed
using Tri-X film with \\ min exposures. These same sections were then stained
with Erlich's haematoxylin and eosin.
RESULTS
FT' The results obtained in these experiments are summarized in Tables 1-5.
Staging of regenerates was according to Freeman (1963).
No regenerates were observed in any of the 30 animals where only the outer
cornea had been wounded (Table 1) and in most of these cases the attachment
between the inner and outer cornea had re-formed. However, where both the
inner and outer corneas had been wounded, lens regeneration had been initiated
in 22 % of cases and had reached stage III at 10 days after the operation (Table
1). The regenerates appeared as epithelial vesicles associated with the inner
cell layer of the outer cornea. Similarly, lens regeneration had been initiated
in 25 % of tadpoles that had undergone lens dislocation (Table 2). In both
types of experiment the regenerates failed to develop beyond stage III, as
evidenced by arrested stage-Ill regenerates in tadpoles sacrificed at 20 days
(Tables 1 and 2 and Fig. 2), and in many cases the inner cornea could be seen
to have re-grown between the regenerate and the eye cup.
Where the lens had been removed and then re-implanted, lens regeneration
had occurred in 57 % of cases by 10 days (Table 3). Stage-Ill regenerates were
found in tadpoles sacrificed at 6 days (Fig. 3) and although some became
arrested at this stage, others continued to develop as evidenced by stage-IV
regenerates in tadpoles sacrificed at 10 days. Such regenerates showed nuclear
and nucleolar enlargement in cells furthest from the cornea, which is characteristic of stage IV and the first sign of lens fibre formation. In these cells a
positive immunofluorescence reaction indicative of lens crystallin synthesis
Lens regeneration from cornea of larval Xenopus
209
20 jum
Fig. 2. Arrested stage-Ill lens regenerate (LR) from a tadpole sacrificed 20 days
after dislocation of the original lens (OL).
Fig. 3. Stage-Ill lens regenerate (LR) from a tadpole sacrificed 6 days after lensectomy and re-implantation of the original lens (OL).
Fig. 4. Stage-IV lens regenerate (LR) stained with F.I.T.C. labelled rabbit antiXenopus laevis total lens protein antibody. The tadpole was sacrificed 10 days
after lensectomy and re-implantation of the original lens (OL).
Fig. 5. Stage-V lens regenerate (LR) from a tadpole sacrificed 20 days after lensectomy and re-implantation of the original lens (OL). Note that unlike the arrested
regenerate shown in Fig. 2, the stage-V regenerate has formed primary lens fibres.
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J. G. REEVE AND A. E. W I L D
Table 1. Corneal wounding
Type of operation
Days after
operation
No. of
operations
10
30
30
10
20
25
25
23
25
Wounding
Outer cornea only
Wounding
Outer cornea
Inner cornea
No. of
No. of
Stage of
recoveries regenerates regeneration
5
4
III
III
Table 2. Lens dislocation
Days after
operation
No. of
operations
No. of
recoveries
No. of
regenerates
Stage of
regeneration
10
20
Total
20
30
50
16
28
44
4
8
12
III
III
Table 3. Lensectomy followed by re-implantation of the lens
Days after
operation
No. of
operations
No. of
recoveries
No. of
regenerates
Stage of
regeneration
6
10
20
10
28
30
68
8
24
24
56
3
13
10
26
III and IV
III* and V
Totals
Ill
* Arrested stage III.
was observed (Fig. 4). Several of the regenerates were mis-shapen, possibly
due to the original lens restricting the space available for growth. Stage-V
regenerates were found in tadpoles sacrificed 20 days after re-implantation
of the lens; these showed primary lens fibre formation and secondary fibre
differentiation (Fig. 5).
As can be seen from Table 4, 58 % of the corneas implanted into the posterior
eye chamber regenerated lenses in the presence of the original lens. Some
implants had developed several foci of lens differentiation, giving rise to multiple
lentoids (Fig. 6), but in others, only a single lentoid had formed (Fig. 7). In
such transplantation experiments lens regeneration was also observed from
the host cornea alone, and, in several cases, from both the host cornea and the
corneal implant. The majority of the regenerates formed from the host cornea
had again been arrested at stage III, although in one case the regenerate had
reached stage V of regeneration in the presence of both the original lens and
a much larger regenerate that had developed from a corneal implant.
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Lens regeneration from cornea of larval Xenopus
Fig. 6. Multiple stage-V lens regenerates (arrows ABC) formed from corneal
implant (CI) in the posterior eye chamber. The tadpole was sacrificed 14 days after
lensectomy and re-implantation of the host lens (HL).
Fig. 7. Single stage-V lens regenerate (LR) formed from a corneal implant in the
posterior eye chamber. The tadpole was sacrificed 14 days after lensectomy and
re-implantation of the host lens (HL).
Table 4. Corneal implant into posterior eye chamber in
presence of re-implanted lens
No. of
operations
No. of
recoveries
Host lens
present
Corneal
implant
present
20
19
18
12
Regeneration
from corneal Stage of
implant regeneration
7
V
Table 5. Control lensectomy
Days after
operation
No. of
operations
No. of
recoveries
No. of
regenerates
Stage of
regeneration
20
50
49
31
V
Histological examination of lenses in eyes which had undergone the operations
described, showed the majority to be normal in appearance. Where lens damage
had occurred, there was no correlation between such damage and incidence
of lens regeneration from the cornea.
In control eyes where permanent lensectomy had been carried out, lens
regeneration from the cornea occurred in 60 % of cases and all regenerates
underwent full lens differentiation to give stage-V lenses (Table 5).
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J. G. REEVE AND A. E. WILD
DISCUSSION
The survival (based on histological criteria) of dislocated and re-implanted
lenses in eyes of larval Xenopus laevis allows for an analysis of any inhibitory
effect which the lens may have on lens regeneration from the cornea. Our
results clearly show that lens regeneration can occur from the cornea even in
the presence of the host lens. Thus in many cases where the lens was removed
and then re-implanted, the cornea was able to undergo full lens differentiation
to give stage-V lenses in the presence of the original lens. Contrary to the
in vitro findings of Manwaring (1972) our results show no real evidence of an
inhibitory lens factor operating in vivo. Whilst arrested regeneration was
observed, this was mainly in the eyes where corneal wounding or lens dislocation had been carried out, and does not necessarily imply the existence of
an inhibitory lens factor, as will be discussed later. Also lens regeneration from
the cornea in the presence of lens did not occur in every case but this finding
is no different from that observed in lens regeneration from the cornea following
permanent removal of the lens, as evidenced from the results obtained by
Freeman (1963) and by our own results for control lensectomized tadpoles.
Efficient wounding of the inner cornea seems to be a prerequisite for lens
regeneration, since wounding of the outer cornea alone failed to initiate it
and re-growth of the inner cornea between the developing regenerate and the
eye cup prevented further development as evidenced by arrested stage-Ill
regenerates.
Lens regeneration in the presence of the lens appeared normal in that those
regenerates attaining full lens differentiation followed the same sequence of
morphological events as seen in control lensectomized eyes. Furthermore, our
immunofluorescence studies confirmed that the regenerates were capable of
synthesizing crystallins in the presence of the lens, the stage of onset and cellular
localization of crystallin synthesis showing close correspondence to that
described by Brahma & McDevitt (1974) following normal lensectomy of
Xenopus tadpoles.
The fact that many regenerates formed from the in situ cornea had been
arrested at stage III, suggests that with time the environment in the anterior
eye region became less able to support regeneration. This is in marked contrast
to the posterior eye chamber, where all regenerates developing from implanted
cornea underwent lens fibre differentiation to give stage-V regenerates.
We believe that the most probable explanation for the finding that lens
regeneration occurs in the presence of the lens is that contrary to the suggestions made by Campbell & Jones (1968) and Manwaring (1972), the lens
in Xenopus tadpoles does not produce an inhibitory factor. Rather we suggest
that a stimulatory factor is released by the eye cup into the posterior eye
chamber which is able to reach the inner cell layer of the outer cornea via the
wounded inner cornea and thus initiate lens regeneration. Access to this factor
Lens regeneration from cornea of larval Xenopus
213
is necessary for the continued development of the regenerating lens. Re-growth
of the inner cornea would isolate developing regenerates from eye cup factor,
which would account for why some became arrested at stage III during
development from in situ cornea. Similarly, the availability of eye cup factor
to the in situ cornea may be reduced by the intervening lens. On the other
hand, ready access to eye cup factor would account for why successful differentiation of lens occurred in cornea transplanted to the posterior eye
chamber, since the cornea in this position would not be exposed to the blocking
action of either the inner cornea or the lens. The relatively higher frequency,
and the more advanced development, of regenerates found in eyes which had
undergone lensectomy and lens re-implantation compared to those that had
undergone corneal wounding or lens dislocation, can be explained in the
same way. Lensectomy and lens re-implantation would more extensively
damage the inner cornea and therefore more greatly delay its repair than
would mere wounding or lens dislocation, thus allowing the outer cornea
a longer exposure to eye cup factor. The blocking action of the lens would
also be less effective in the case of the re-implanted lens since unlike lens
dislocation lensectomy would completely destroy the connectives between the
iris and the lens. In this context, the slight increase in the width of the pupillary
space caused during lens re-implantation, would also be an important factor.
Experiments designed to evaluate the role of the retina as a source of controlling
factor, and to more precisely determine the nature of the barrier presented
by the inner cornea, are at present being carried out in our laboratory.
Similar experiments using re-implanted lens to assess the effect of the lens
on lens regeneration were made by Dinnean (1942) on Taricha torosus larvae
and by Eguchi (1961) on Triturus larvae, species in which lens regeneration
occurs from the dorsal iris. Dinnean found that lens regeneration was completely inhibited by the presence of normal or even vacuolated lenses, whilst
Eguchi found lens regeneration did occur in the presence of lens but only if
the dorsal iris did not make contact with it. The difference between the present
observations on corneal lens regeneration and those of Dinnean (1942) and
Eguchi (1961) on Wolffian lens rengeration must reflect fundamental differences
in the control of lens regeneration in the two systems.
Julie G. Reeve was supported by a Science Research Council post-graduate training
award for which grateful acknowledgement is made.
REFERENCES
A. E. (1975). Practical Studies of Animal Development. London:
F. S. & WILD,
Chapman and Hall.
BRAHMA, S. K. & MCDEVITT, D. S. (1974). Ontogeny and localization of the lens crystallins
in Xenopus laevis lens regeneration. /. Embryol. exp. Morph. 32, 783-794.
CAMPBELL, J. C. & JONES, K. W. (1968). The in vitro development of lens from cornea of
larval Xenopus laevis. Devi Biol. 17, 1-15.
BILLETT,
214
J. G. REEVE AND A. E. WILD
DINNEAN, F.
L. (1942). Lens regeneration from the iris and its inhibition by lens re-implantation
in Tritunis torosus larvae. J. exp. Zool. 90, 461-478.
EGUCHI, G. (1961). The inhibitory effect of the injured and the displaced lens on the lens
formation of Tritunis larvae, Embryologia 6, 13-35.
FREEMAN, G. (1963). Lens regeneration from the cornea in Xenopus laevis. J. exp. Zool.
154, 39-65.
MANWARING, G. M. A. (1972). Biochemical studies on lens induction. Ph.D. thesis, University
of Edinburgh.
REEVE, J. G. & WILD, A. E. (1977). Lens regeneration from cornea in tail ectopic eyes of
Xenopus laevis tadpoles. Experientia 33, 1210-1211.
SAINTE-MARIE, G. (1962). A paraffin embedding technique for studies employing immunofluorescence. /. Histochem. Cytochem. 10, 250-256.
STONE, L. S. & VULTEE, J. H. (1949). Inhibition and release of lens regeneration in the
dorsal iris of Triturus viridescens. Anat. Rec. 103, 560.
STONE, L. S. (1965). The regeneration of the crystalline lens. Invest. Opthal. 4, 420-432.
WAGGONER, P. (1973). Lens differentiation from the cornea following lens extirpation or
cornea transplantation in Xenopus laevis. J. exp. Zool. 186, 97-110.
(Received 30 May 1978)