/. Embryol. exp. Morph. Vol. 32, 3, pp. 783-794, 1974
783
Printed in Great Britain
Ontogeny and localization of the lens crystallins
in Xenopus laevis lens regeneration
By SAMIR K. BRAHMA 1 AND DAVID S. McDEVITT 2
From the Department of Medical Anatomy and
Embryology, The State University, Utrecht, and the
Department of Animal Biology, University of Pennsylvania
SUMMARY
Ontogeny and localization of the lens crystallins, especially the y-crystallins were investigated in Xenopus laevis lens regenerating system by the 'indirect' immunofluorescence
staining method. Antibodies directed against Rana pipiens y-crystallin antigen were used for
the detection of this crystallin; the validity of such an experiment has been shown in a
previous report. To detect total lens proteins we used X. laevis anti-total lens protein antibody.
The regenerates were staged according to Freeman (1963) and the first positive reaction
with both the two antisera was observed in an early stage-4 regenerate. The site of the
immunofluorescence reaction was nearly identical in both, suggesting that y-crystallins are
one of the first, if not the first of the lens crystallins to appear during lens regeneration.
The secondary fibres, when developed, showed less immunofluorescence than the primary
fibres with R. pipiens anti-y crystallin antibody, though the reaction was intense in the
secondary fibres with X. laevis anti-total lens protein antibody.
The intensity and distribution of immunofluorescence increased with the growth of the
lens. With the R. pipiens anti-y crystallin antibody, the lens epithelium did not show any
immunofluorescence reaction at any stage of lens regeneration. With X. laevis anti-total
lens protein antibody, the epithelium showed an immunofluorescence reaction earlier than
in the normal lens development. With the two antisera we used, we did not observe any
immunofluorescence outside the lens tissue.
INTRODUCTION
Lens crystallins of various amphibian species are classified into a-, /?-, and
y-crystallins (McDevitt, 1967; Campbell, Clayton & Truman, 1968; Brahma
& van Doorenmaalen, 1969; Polansky & Bennett, 1970; Brahma & Bours,
1971; McDevitt, 1972; Polansky & Bennett, 1973). Biochemical and immunofluorescence studies have shown that in bovine and amphibian lenses, the
y-crystallins can be detected only in fibre cells or in those cells which are in
the process of developing into fibres and have entered into a permanent
stationary phase. These cells thus become biochemically specialized for the
1
Author's address: Department of Medical Anatomy and Embryology, The State
University, Janskerkhof 3A, Utrecht, The Netherlands.
2
Author's address: Department of Animal Biology, School of Veterinary Medicine,
University of Pennsylvania 19174, U.S.A.
50-2
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S. K. BRAHMA AND D. S. MCDEVITT
synthesis of tissue specific macromolecules, the y-crystallins (Papaconstantinou,
1965; Takata, Albright & Yamada, 1965; McDevitt, Meza & Yamada, 1969;
McDevitt & Brahma, 1973).
In most of the urodele amphibian species it has been shown that lens
regeneration takes place from the dorsal iris all through the life of the animal
(Reyer, 1954; Stone, 1959; Reyer, 1962); while in the South African clawed
toad, Xenopus laevis, an anuran amphibian, lens regeneration has been reported
to take place from the corneal epithelium only, and to be restricted to the
larval stages (Overton & Freeman, 1960; Freeman & Overton, 1961; Freeman,
1963; Waggoner, 1973). However, Campbell (1963) reported lens regeneration
from dorsal iris in adult X. laevis although Brahma & van Doorenmaalen
(1968) failed to observe lens regeneration from dorsal iris in adults of the
same species.
Campbell (1965) made immunofluorescence studies of the total lens crystallin
antigens in a X. laevis lens regenerating system by the 'direct' method; but
no reports are available on the ontogeny of the y-crystallins during lens
regeneration in this species, though experiments have been described on the
ontogeny and localization of y-crystallins during lens regeneration in urodeles
and in the normal lens development of R. pipiens and X. laevis (Takata et al.
1964a, b, 1965, 1966; McDevitt et al 1969; McDevitt & Brahma, 1973).
In this article results are presented regarding the ontogeny and localization
of lens crystallins, especially the y-crystallins, in the X. laevis lens regenerating
system, studied by the 'indirect' immunofluorescence staining method.
We used X. laevis anti-total lens protein antibodies for the detection of
total lens crystallins and R. pipiens anti-y crystallin antibodies for the detection
of y-crystallins. The validity of using heterologous anti-y crystallin antibody
against X. laevis lens has been confirmed previously (McDevitt & Brahma,
1973). Moreover, it has recently been shown that R. pipiens anti-y crystallin
antibody can be used successfully to detect y-crystallin antigens in newt lens
regenerates both in vivo and in vitro (Nothiger, McDevitt & Yamada, 1971;
Yamada, Reese & McDevitt, 1973).
MATERIALS AND METHODS
The collection and rearing of fertilized eggs from X. laevis up to larval
stages has already been described (McDevitt & Brahma, 1973). When the
larvae reached stages between 52 and 54 (Nieuwkoop & Faber, 1956) they
were used in the present experiment. From any single clutch, larvae with
identical growth rates were selected for the operation described below.
Before the operation, the larvae were anaesthetized with MS 222 as suggested
by Freeman (1963). They were transferred to a Petri dish with a base of
3 % agar and containing conditioned tap water. A groove was cut in the agar
base to hold the animals in position during the operation. Lenses were removed
from the left eye with sharp tungsten needles under a dissecting binocular
Crystallins in Xenopus laevis lens regeneration
785
microscope, and each lens after lentectomy was carefully examined under the
microscope, with high magnification, to check the completeness of removal.
Then the larvae were returned immediately to an aquarium with constant
aeration.
Animals with regenerates from 2nd day till 18th day after lens removal
were anaesthetized, decapitated and the heads washed in water at 4 °C. The
heads were fixed either in cooled Carnoy (absolute alcohol:chloroform:glacial
acetic acid, 6:3:1) or in 9 5 % ethanol at 4 °C, processed and sectioned at
5 /<m thickness according to McDevitt et al. (1969).
Regenerates were stained according to the methods described earlier (McDevitt et al. 1969; McDevitt & Brahma, 1973) and identified according to the
five stages of Freeman (1963). The antibodies used in the present series were
from the same stock used in our previous experiment (McDevitt & Brahma,
1973). The X. laevis anti-total lens protein antibody was diluted to remove
any non-specific staining (Goldman, 1968); this was not necessary with the
anti-y crystallin antibody.
After staining, sections were mounted in 'Mowiol' N-30-38 extra (Hoechest
AG, Frankfurt/M, Germany) according to the methods of Thomason & Cowart
(1967). Lenses were examined with an E. Leitz fluorescence microscope and
photographed under identical conditions to those described previously (Brahma
6 van Doorenmaalen, 1971; Brahma, Rabaey & van Doorenmaalen, 1972).
From the same regenerate, phase contrast pictures were taken to demonstrate
the site of the immunofluorescence.
Staging of the lens regenerates
Freeman (1963) described the process of lens regeneration from the cornea
in X. laevis larvae in detail and he divided the entire process into five arbitary
stages. A running summary of all these stages as described by Freeman (1963)
is presented here to facilitate understanding of the stages mentioned in this
article.
Stage 1. This is the stage in which, immediately after lens removal, cells
of the inner layer of the outer cornea change from squamous to cuboidal
shape.
Stage 2. The cells of the inner layer of the outer cornea at this stage form a
mass, two to three cell layers thick, in the pupilary space.
Stage 3. The loose cells of the aggregate become oriented with regard to
each other and tend to separate from other corneal cells and form a rudiment
which grows in size into the space occupied by the original lens.
Stage 4. At the beginning of this stage some of the cells farthest from the
cornea show enlargement of their nuclei and nucleoli. Mitotic activities are
restricted to the periphery of the vesicle and cells with large nuclei produce
irregular fibres, the first sign of fibre formation. From this stage, the regenerate
is generally found to be separated from the cornea.
786
S. K. BRAHMA AND D. S. MCDEVITT
Crystallins in Xenopus laevis lens regeneration
787
Stage 5. The secondary fibres begin to form the equatorial zone of the lens
epithelium and the nuclei of the primary fibres begin to disappear. From this
stage onwards, no major histological changes are observed in the lens except
that it continues to grow in size.
Freeman (1963) also sub-divided some of the stages i.e. 3, 4 and 5 into early,
middle and late stages for a more precise categorization of the process of lens
regeneration.
RESULTS
Immunofluorescence with anti-total lens protein antibody
The first positive immunofluorescence reaction observed under our experimental conditions was at early stage 4. It was found to be localized in a
number of cells in an area of the lens rudiment farthest from the cornea, where
future lens fibres would develop (Fig. 1). With further differentiation of the
regenerate, more and more cells of the primary fibre cell area showed positive
immunofluorescence and in the mid stage-4 regenerate, the external layer of
the vesicle started to show weak immunofluorescence (Fig. 2). By early stage 5
when secondary fibres began to develop from the equatorial zone, both primary
and secondary fibres showed more intense immunofluorescence than the
epithelium (Fig. 3). In the mid stage-5 regenerate the secondary fibres showed
more fluorescence than the primary fibres, and the epithelium showed less
fluorescence than both the primary and secondary fibres. But in general,
intensity of immunofluorescence reaction was enhanced (Fig. 4).
From this stage on, the lens did not show any major structural change,
except that it increased in size and the intensity of immunofluorescence reaction
was increased throughout the area of the lens.
Figs. 5, 6, 7 and 8 are the phase contrast pictures of the tissues shown in
Figs. 1, 2, 3 and 4.
Fig. 1. Immunofluorescence photomicrograph of an early stage-4 regenerate exposed
to X. laevis anti-total lens protein antibody x281. The regenerate was fixed in
cooled Carnoy.
Fig. 2. Immunofluorescence photomicrograph of a mid stage-4 regenerate exposed
to X. laevis anti-total lens protein antibody x 243. The regenerate was fixed in
cooled 95% ethanol.
Fig. 3. Immunofluorescence photomicrograph of an early stage-5 regenerate exposed
to X. laevis anti-total lens protein antibody x 206. The regenerate was fixed in
cooled 95 % ethanol.
Fig. 4. Immunofluorescence photomicrograph of a mid stage-5 regenerate exposed to
X. laevis anti-total lens protein antibody x 172. The regenerate was fixed in cooled
Carnoy.
Figs. 1-4 are dark field photomicrographs.
Figs. 5-8 are the phase contrast photomicrographs of the above lenses with
identical magnifications and shown as histological reference.
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S. K. BRAHMA AND D. S. MCDEVITT
Crystallins in Xenopus laevis lens regeneration
789
Immunofluorescence with anti-y crystallin antibody
With this antiserum we also observed that the first positive immunofluorescence
reaction appeared in an early stage-4 regenerate. It was localized at approximately the same area which showed the earliest positive reaction with X. laevis
anti-total lens protein antibody (Fig. 9). As the cells in this region started to
differentiate into fibres, more and more cells became involved in the synthesis
of this crystallin. In the mid stage-4 regenerate it became quite evident that
only fibre cells were involved in the synthesis of this crystallin class, and no
reaction could be observed in the external layer of the vesicle which would
ultimately develop into lens epithelium (Fig. 10). In the early stage-5 regenerate,
the secondary fibres were almost negative, while the fluorescence was restricted
only to the primary fibres (Fig. 11). In the mid stage-5 regenerate, the secondary
fibres showed fluorescence, though less than primary fibres, while the total
pattern of intensity of immunofluorescence increased (Fig. 12). This state of
fluorescence prevailed in regenerates from mid stage-5 regenerate onwards. In
no case could we detect immunofluorescence in the lens epithelium, and no
immunofluorescence in the cornea was observed with either antibody.
Figs. 13, 14, 15 and 16 are phase contrast pictures of the tissues shown in
Figs. 9, 10, 11 and 12.
In a few cases we observed double lenses and in one case multiple lens
formation. Two such regenerates were treated with anti-y crystallin antibody.
It was observed that the stages of differentiation of the double lenses were
identical, and their immunofluorescence patterns were also identical (Fig. 17),
while in the other with multiple lenses, only one showed fibre differentiation
and the expected positive immunofluorescence reaction with anti-y crystallin
antibody (Fig. 18).
Figs. 19 and 20 are phase contrast pictures of the tissues shown in Figs. 17
and 18.
Fig. 9. Immunofluorescence photomicrograph of an early stage-4 regenerate
exposed to R. pipiens anti-y crystallin antibody x 271. The regenerate was fixed in
cooled Carnoy.
Fig. 10. Immunofluorescence photomicrograph of a mid stage-4 regenerate exposed
to R. pipiens anti-y crystallin antibody x 234. The regenerate was fixed in cooled
95%ethanol.
Fig. 11. Immunofluorescence photomicrograph of an early stage-5 regenerate
exposed to R. pipiens anti-y crystallin antibody x 199. The regenerate was fixed
in cooled 95% ethanol.
Fig. 12. Immunofluorescence photomicrograph of a mid stage-5 regenerate exposed
to R. pipiens anti-y crystallin antibody x 164. The regenerate was fixed in cooled
Carnoy.
Figs. 9-12 are dark field photomicrographs.
Figs. 13-16 are the phase contrast photomicrographs of the above lenses with
identical magnifications and shown as histological reference.
790
S. K. BRAHMA AND D. S. MCDEVITT
Fig. 17. Immunofluorescence photomicrograph of a regenerate showing double
lens formation and exposed to R. pipiens anti-y crystallin antibody. Both the lenses
are in same stage of differentiation and show equal intensity of immunofluorescence
x .166. The regenerate was fixed in cooled Carnoy.
Fig. 18. Immunofluorescence photomicrograph of a regenerate showing multiple
lens formation and exposed to R. pipiens anti-y crystallin antibody. Only one with
fibre differentiation shows positive reaction x 192. The regenerate was fixed in
cooled Carnoy.
Figs. 17 and 18 are dark field photomicrographs.
Figs. 19 and 20 are the phase contrast photomicrographs of the above lenses with
identical magnifications and shown as histological references.
Reaction with serum from non-immunized rabbits
In this series we determined the specificities of the immune antibody types
by replacing them with serum from non-immunized rabbits. Immunofluorescence
reaction in both cases were negative, indicating specificities for total lens and lens
fibre cells (anti-total lens protein and anti-y crystallin antibodies, respectively).
DISCUSSION
Campbell (1965), with his unabsorbed X. laevis anti-total lens protein antiserum, found a positive immunofluorescence reaction in the corneal epithelium
before the lens regenerate was developed and this situation persisted till the
Crystallins in Xenopus laevis lens regeneration
791
regenerate reached stage 5. He observed positive immunofluorescence in the
regenerate from stage 3; in late stage 3 its intensity was increased and some
cells of the posterior margin of the lens vesicle showed strong fluorescence.
In the present series of experiments with X. laevis anti-total lens protein
antibody made specific by dilution, we observed immunofluorescence only
from the early stage-4 regenerate and not in the cornea.
Campbell (1965) also reported that with his antiserum he did not observe
any immunofluorescence in the lens epithelium of the regenerates and he
suggested that 'most lens antigens are associated with fibres'. With our antibody (total lens protein) we, on the other hand, could detect positive immunofluorescence in the epithelium, and, in contrast to the situation in X. laevis
normal lens development (McDevitt & Brahma, 1973), the fluorescence appeared
quite early during lens regeneration. This difference between the present
observations and those of Campbell (1965) could be due to differences in the
specificities of the antisera and/or the techniques employed.
The appearance of y-crystallins in the prospective fibre cells as they begin
to elongate is well established in urodele regenerates both in vivo and in vitro;
also in normal anuran and mammalian lens development (Takata et al. 1966;
McDevitt et al. 1969; Yamada et al. 1973; Schubert, Trevithick & Hollenberg,
1970).
The results with X. laevis lens regenerates differ from the above in the
presence of a positive immunofluorescence reaction with R. pipiens y-crystallin
antibody in the prospective fibres cells earlier in the histogenesis of the
regenerate. Similar results have also been recorded in X. laevis normal lens
development with R. pipiens anti-y-crystallin antibody (McDevitt & Brahma,
1973), thus reinforcing the concept of X. laevis as an organism with aberrant
lens differentiation. In both cases, y-crystallins are one of the first, if not
the first, of the lens crystallins to appear.
McDevitt (1972) reported that in R. pipiens normal lens development, both
/?- and y-crystallins, could be detected simultaneously as the first to appear by
classical immunological methods. Yamada (1967) reported in his review article
that in the newt lens regenerate, a-, and /?-crystallins appear first in the cells
before they enter the phase of fibre differentiation; and as soon as these cells
enter the terminal cell cycle, y-crystallins appear. This difference could be a
species difference or a difference in the histogenesis of the lens regenerates.
In addition, as stated by Yamada (1966) results obtained with his a- and
/?-crystallin antisera should be evaluated with reservations because of the
antigen contamination he reported.
The importance of y-crystallins in relation to fibre differentiation is once
again demonstrated in our experiments. However, some difference could be
observed between lens regeneration in urodeles and X. laevis. In the former,
the lens regenerates from the dorsal iris after lentectomy, though it can also
develop from the ventral iris after the administration of TV-methyl-TV'-nitro-iV-
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S. K. BRAHMA AND D. S. MCDEVITT
nitrosoguanidine (Eguchi & Watanabe, 1973), and these are known to be the
cases of metaplastic changes since the iris is already a differentiated tissue, and
all epithelial cells are terminally differentiated (Yamada, 1972). In X. laevis,
however, lens regeneration appears to be restricted to larval stages and to
come only from the corneal cells. At these stages, the development of the
cornea is not complete (Freeman, 1963) and therefore, this does not appear
to be a case of metaplastic change.
Amongst the vertebrates, either a normally developing lens or a lens regenerate pass through a typical vesicle stage where the external cell layer that
will give rise to the lens epithelium can be distinguished from internal cell
layer that would develop into primary fibres. In X. laevis, this vesicle is shortlived both in normal lens development (McDevitt & Brahma, 1973) and also
in the regenerates. In addition, there is no such clear cut difference between
external and internal layers in normal X. laevis development, and the vesicle
stage appears after fibre formation. Moreover, reaction to anti-y crystallin
antibody appears long before any sign of fibre formation and in this respect,
X. laevis lens development, both normal and regenerating, differs from that of
lenses of other amphibian species studied so far. The developmental cause for
such deviation is not known.
There are also other differences between corneal regeneration and normal
lens development in X. laevis. In the former, the lens vesicle appears much
earlier and the epithelium shows a positive immunofluorescence reaction with
anti-total lens protein antibody earlier than in normal lens development,
indicating a comparatively precocious synthesis of a- and/or /?-crystallins in
this cell type.
In addition, results from multiple lens regenerates, the occurrence of which
ensures an identical milieu for differentiation, show strikingly that y-crystallins
are associated only with the process of fibre differentiation.
We thank Professor W. J. van Doorenmaalen for valuable discussions and to Mr T. H.
Hulskes for the illustrations. The research was partly supported by N.S.F.-I.G. 73-25 to
the University of Pennsylvania (D.S.McD).
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(Received 29 March 1974)