/. Embryo/, exp. Morph. Vol. 44, pp. 149-165, 1978
Printed in Great Britain © Company of Biologists Limited 1978
149
Cell division, cell elongation and distribution of
a-, (3- and y-crystallins in the rat lens
By J. W. McAYOY
From the Nuffield Laboratory of Ophthalmology, University of Oxford
SUMMARY
The vertebrate lens comprises cells from only one cell lineage and these exist in two
distinct forms. Fibre cells make up the bulk of the lens and a monolayer of epithelial cells
covers the anterior surface of the fibres. A quantitative analysis of cell division and cell
specialization (elongation) in the lenses of 1-day-old and 6- to 7-week-old rats showed that
the rat lens can be separated into two compartments; a proliferation compartment in the
epithelium of the anterior lens and an elongation compartment in the posterior lens. In 6to 7-week-old rats the proliferation compartment is divided into three sub-compartments
which appear to be related to the anatomy of the anterior eye. Lens epithelial cells lying in the
vicinity of the ciliary body have an average cell cycle time of 7-2 days, epithelial cells which
lie below the iris have an average cell cycle time of 32-3 days, and cells in the central epithelium divide most slowly. The epithelium is bathed for the most part by the aqueous
humour and the posterior part of the lens is surrounded by the vitreous humour. From this
analysis as well as evidence from other laboratories it is suggested that a proliferation
factor(s) may be present in the aqueous and that an elongation factor(s) which comes from
the retina may be present in the vitreous.
There are three major groups of lens specific proteins, a-, /?- and y-crystallins. These
proteins were separated by gel chromatography and antibodies raised against them in
rabbits. Alpha-, /?- and y-crystallins were localized in sections of the 1-day-old rat lens by
the indirect immunofluorescence method. Epithelial cells in the proliferation compartment
contained only a-crystallin but all three groups of crystallins were detected in cells of the
elongation compartment. /?-Crystallin was detected first in elongating cells that had stopped
dividing and y-crystallin was detected later in the elongation process. Thus, synthesis of
fi- and y-crystallins may normally depend on the elongation factor(s) from the retina. Also,
since there is a close relationship between cessation of cell division and detection of /?crystallin, another factor governing the synthesis of ^-crystallin may be whether the cells
have ceased dividing. In this way the expression of crystallin genes may be co-ordinated as
the lens grows and cells make the transition from proliferation to elongation compartments.
INTRODUCTION
The development of tissue structures and the maintenance of characteristic
growth patterns within tissues are important areas for study in developmental
biology. The vertebrate lens is a relatively simple tissue in the sense that it is
made up of cells from only one cell lineage; blood, connective tissue or nerve
cells are not present. Lens cells exist in two distinct forms; fibre cells make up
1
Author's address: University of Oxford, Nuffield Laboratory of Ophthalmology, Oxford
0X2 6AW, U.K.
150
J. W. McAVOY
the bulk of the lens and a monolayer of epithelial cells covers the anterior
surface of the fibres (Fig. 1). The epithelial layer is bathed for the most part by
aqueous humour and the posterior part of the lens is surrounded by vitreous
humour. Essentially lens growth depends on division of epithelial cells and
specialization of some of their progeny into fibre cells (Hanna & O'Brien, 1961).
Although these processes are known to occur during lens growth little is known
about their control. Considerable analysis has been carried out on the control
of cell division in the injured and cultured lens, mainly in frogs (see Harding,
Reddan, Unakar & Bagchi, 1971, for review), but little is known about control
of cell division in the normal lens. Moreover, and rather surprisingly, there is a
lack of precise information on how cell division in the lens is spatially related
to fibre cell formation and how both these processes are related to the two
different lens environments, i.e. aqueous and vitreous.
This work reports how the processes of cell division and cell specialization
are spatially related in 1-day-old and 6- to 7-week-old rat lenses and suggests
how these processes may be governed by the anterior and posterior lens
environments respectively. In addition, the three groups of lens specific proteins,
a-, /?- and y-crystallins were localized in 1-day-old lenses by immunofluorescence
so that the molecular basis of structural and functional changes involved in the
transition of epithelial cells into fibre cells during lens growth could be analysed.
This work forms part of a study aimed at elucidating the general mechanisms
involved in lens morphogenesis and in its maintenance during growth.
MATERIALS AND METHODS
Quantitative studies on cell division and cell elongation
Cell division and cell elongation were analysed in different positions in the
lens epithelium. For this purpose cells were assigned positional numbers using
a somewhat similar system to that of Mikulicich & Young (1963). In sagittal
sections, of the cells at the equator of the lens, the last cell in the epithelium
with a longitudinal axis still at right angles to the epithelium/fibre junction
occupied position 1 (see Fig. 1). Cells were numbered consecutively in both
anterior and posterior directions, those in a posterior direction being given
negative signs ( - 1 ) , ( - 2 ) , ( - 3 ) etc.
Distribution of dividing cells
Patterns of cell division were analysed in lenses of 1-day-old and 6- to 7-weekold Wistar albino rats killed at 12.00 noon.
(a) 6- to 7-week-old rats. From these older rat lenses whole mounts of the
epithelium were prepared according to the method of Howard (1952). Thus, in
a single preparation all the mitotic cells in one lens were present. In a total of
seven whole mounts, mitotic cells were scored according to cell positions
equivalent to those in histological sections.
Growth of the rat lens
151
Proliferation
compartment
ON
Fig. 1. Schematic diagram of rat eye; Co, cornea; Ca, capsule; I, iris; S, lens
sutures; PC, posterior chamber; AC, anterior chamber; CP, ciliary process; N, lens
nucleus; Z, zonular fibres; VH, vitreous humour; R, retina; ON, optic nerve.
Proliferation sub-compartments are demarcated. Sub-compartment 1 (SC .1) from
about cell position 33-68 has an average mitotic index of 0-27%. Sub-compartment
2 (SC 2) from about cell position 69-150 has an average mitotic index of 009%
and sub-compartment 3 (SC 3) from about cell positions 151-200 has an average
mitotic index of 002%. Inset shows cell position 1 is the last cell in the epithelium
(E) with a longitudinal axis at 90° to the junction between epithelium andfibres(F).
Cells are numbered from here towards the centre of the epithelium. Numbering in
the opposite direction the cells are given negative signs.
(b) 1-day-old rats. Whole mounts of these smaller lenses could not be prepared and instead the distribution of dividing cells was indicated by [3H]thymidine autoradiography. Six rats were each given an intra-peritoneal
injection of 10 /tCi/g body weight of methyl-[3H]thymidine (19 Ci/mmole,
The Radiochemical Centre, Amersham) and killed with ether after \\ h. One
whole eye from each rat was fixed in Carnoy's (3:1 ethanol/acid), embedded in
paraffin and sagittal sections cut at 4/tm. Autoradiography was carried out as
described previously (McAvoy & Dixon, 1978). After staining with haematoxylin, cells with labelled nuclei were scored according to their position.
Cell cycle analyses
Estimates of average cell cycle times in different parts of the lens were based
on the technique of Puck & Steffen (1963). They predicted that for exponentially
152
J. W. McAVOY
0014
0012
| 0010
'% 0 008
+ 0 006
o
>-> 0-004
0002
0
2
4
6
8
Time (h)
Fig. 2. Mitosis collection function for proliferation sub-compartments 1 ( • ) and 2 (#).
growing asynchronous cells the logarithm of one plus the mitotic index is
proportional to the time for which mitoses had been collected using a metaphase
blocking agent. The relationship between mitotic index and time is referred to
as the mitosis collection function. Cell cycle time (T) may be calculated using the
formula T = 0-3010/6, where b is the gradient of the mitosis collection function.
In this study vincristine sulphate ('Oncovin', Eli Lilly & Co. Ltd, Basingstoke)
was used as the metaphase blocking agent. Six- to 7-week-old rats were given
sub-conjunctival injections of approximately 25 /A vincristine sulphate (2 mg/
1 ml) in one eye at 9.00 a.m. and killed after 2, 4, 6 or 8 h. Whole mounts were
prepared and carefully examined for anaphase and telophase cells: when these
were observed the whole mount was discarded. In a total of four whole mounts
of each incubation time mitotic cells were scored according to cell position. In
this study mitosis collection functions were plotted for cells in proliferation
sub-compartments 1 and 2 (Fig. 2, see also Fig. 1) and estimates of average cell
cycle times were calculated.
Cell elongation
The heights of epithelial cells and elongating fibre cells at different positions
in both 1-day-old and 6- to 7-week-old rat lenses were measured in sagittal
sections.
Separation of Jens proteins
In mammals there are three major groups of lens specific proteins, a-, /?- and
y-crystallins (see Clayton, 1974; Harding & Dilley, 1976, for reviews). Lenses
from 6- to 7-week-old rats were homogenized in 2-5 ml 0-01 M tris-HCl buffer
Growth of the rat lens
153
(pH 7-3) and lens proteins from the soluble fraction were separated by agarose
gel chromatography according to the method of van Kleef & Hoenders (1973).
Fractions were collected every 16 min and absorbance was measured at 280 nm
on a Pye Unicam spectrophotometer. Fractions were pooled from each of the
a-, /?- and y-crystallin regions, dialysed against water and freeze dried. The
separation was carried out using seven lenses from 6- to 7-week-old rats. To
characterize the separated crystallins, they were electrophoresed on SDSpolyacrylamide gels according to Weber, Pringle & Osborn (1972). Two
separations were carried out. In the first separation, a-crystallin showed two
bands with mobilities of 0-65 and 0-83, ytf-crystallin also showed two bands with
mobilities of 0-60 and 0-66 and y-crystallin one band with a mobility of 0-71.
In the second separation the mobilities were: 0-69 and 0-72 for a-crystallin,
0-60 and 0-63 for /?-crystallin and 0-73 for y-crystallin.
Preparation of antibodies
Antibodies directed against y-crystallin from the first protein separation and
a-crystallin and /?-crystallin from the second separation were raised in 2-3 kg
rabbits. Three rabbits were each initially given a subcutaneous injection of a
1 ml emulsion made up of a-, /?- or y-crystallin solution (1 mg/0-5 ml phosphate
buflFered saline) and Freund's complete adjuvant (Difco) on two sites between
the shoulder blades. After this two different protocols were used. The rabbit
injected with y-crystallin was given three further injections of y-crystallin in
Freund's incomplete adjuvant (Difco), the first two at intervals of 1 week and
the last after an interval of 3 weeks. One week after the last injection it was bled
from the ear vein and the serum collected. Bleeding was carried out once a
week for 8 weeks with an injection of protein and incomplete adjuvant every
third week. Using the second protocol, the two rabbits injected with either aor /?-crystallins were given injections of their respective protein and complete
adjuvant mixture every third week. One week after the third injection the
rabbits were bled and serum collected. Bleeding was continued at weekly
intervals for 8 weeks.
Immunodiffusion and immunoelectrophoresis
The specificity of antisera was tested by immunoelectrophoresis according to
the method described by Clausen (1969). Anti a-, anti /?- and anti y-crystallins
were run against a-, /?- and y-crystallins and whole lens extract (soluble fraction
of 0-21 g of whole lens homogenized in 5 ml 0-01 M tris-HCl buffer pH 7-3);
anti a- formed only one precipitin band against a-crystallin and whole lens but
did not react with /?- or y-crystallins, anti /?- formed two and sometimes three
bands against /?-crystallin and whole lens but did not react with a- or ycrystallins, anti y- formed two bands against y-crystallin and whole lens but
did not react with a- or /?-crystallins (Fig. 3). Double diffusion by the
Ouchterlony (1953) method showed similar results and it was concluded that
154
J. W. McAVOY
1 mm
Fig. 3. Immunoelectrophoresis slides of a- (upper well), /?- (middle well), and
y- (lower well) crystallins against: (a) anti a-serum in both troughs, a precipitin
band forms with a-crystallin but not with /?- or y-crystallins; (b) anti /?-serum in
both troughs, two precipitin bands form with /?-crystallin but none with a- or
y-crystallins; (c) anti y-serum in both troughs, two precipitin bands form with
y-crystallin but none with a- or /?-crystallins. Slides were stained with naphthalene
black.
within the limits of detection of these methods the anti a-, anti /?- and anti
y-antibodies were specific for a-, /?- and y-crystallins respectively.
Immunofluorescence
The anti-crystallin antibodies were used to localize a-, /?- and y-crystallins
in sections of eyes by the indirect immunofluorescence technique of Weller &
Coons (1954). Eyes were fixed in Carnoy's at 4 °C and were processed for
immunofluorescence by the method of Sainte-Marie (1962). Antibodies were
applied to 4/tm sagittal sections followed by goat anti-rabbit gamma globulin
antibody conjugated with fluorescein isothiocyanate (FITC, Nordic Immunological Laboratories, The Netherlands). To determine the specificity of the
fluorescence the following two controls were carried out. Serum from nonimmunized rabbits was substituted for specific anti-crystallin antisera. The
other control was simply to apply FITC directly to the section without prior
incubation in serum. In both controls no fluorescence was observed.
Crystallins were localized in 1-day-old but not in 6- to 7-week-old rat lenses.
Because 6- to 7-week-old rat lenses are hard and brittle after processing for
Growth of the rat lens
155
histology the cut face of the block needs to be softened in water before sections
can be cut. This treatment results in diffusion of proteins and thus prohibits the
true localization of proteins in these lenses by immunofluorescence.
RESULTS
Patterns of cell proliferation
(a) 6- to 7-week-old rats. Cell division in the lens is restricted to the monolayer
of epithelial cells that covers the anterior lens (Fig. 1). The distribution of
mitotic cells in relation to cell position in the epithelial whole mounts of 6- to
7-week-old rats are shown in Fig. 4. Mitotic figures are not found up to cell
position 17. There is a sharp increase in the mitotic index from 0 to 0-37 %
from positions 17 to 48, followed by a sharp drop to 0-13 % at position 80 and
a slight drop to 0-07 % at cell position 140. There is a further drop in mitotic
index to 0-02 % by position 160, then a plateau up to position 200 followed by
a rise to 0-06 % at the centre of the epithelium. This distribution of mitotic
cells is somewhat similar to the distribution of DNA synthesizing cells in the
rat epithelium recorded by Harding, Hughes, Bond & Schork (1960).
The results reported here indicate three sub-compartments within the proliferation compartment and these appear to be related to the anatomy of the
anterior eye. Sub-compartment 1 extends approximately from cell positions 33
to 68 and lies in the vicinity of the anterior part of the ciliary process (Figs. 1
and 4). The average mitotic index is 0-27 % and the average cell cycle time as
established from the mitosis collection function (Fig. 2) is 7-2 days. Subcompartment 2 extends approximately from cell positions 68 to 150 and is
covered for the most part by the iris (Figs. 1 and 4). The average mitotic index
is 0-09 % and the average cell cycle time is 32-3 days. Sub-compartment 3
extends approximately from cell positions 151 to 200 and is not covered by
any part of the uvea (ciliary body and iris; Figs. 1 and 4). The average mitotic
index is 0-02 %.
Also using a mitotic arrest method von Sallmann, Grimes & McElvain (1962)
calculated cell cycle times of 19 and 31 days respectively for equatorial and
pre-equatorial zones in the lens epithelium of 7- to 8-week-old rats. These
zones appear to correspond roughly to sub-compartments 1 and 2 and the
average cell cycle time in the latter of 32-3 days is in good agreement with 31 days
for the corresponding pre-equatorial zone. However, there is a discrepancy
between 7-2 days and 19 days as calculated for sub-compartment 1 and the
pre-equatorial zone respectively. An explanation for the longer cell cycle time
reported by von Sallmann et al. may be that the non-dividing cells in the first
17 cell positions (see Fig. 4) were included in the equatorial zone.
(b) 1-day-old rats. The distribution of cells labelled with [3H]thymidine is
shown in Fig. 5. No labelled cells are found up to position ( - 5 ) . The greatest
proportion of labelled cells is found in the epithelium between cell positions 5
156
J. W. McAVOY
Cell position 17
Elongation
compartment
Anterior border of
ciliary process
01 -
-5
Lens equator
1
^
-
^
^
100
120
Cell position
140
160
180
200
•
Centre of epithelium
Fig. 4. Patterns of mitotic activity (#) and cell elongation ( • ) from lens equator
to the centre of the epithelium in 6- to 7-week-old rats. The positions of the anterior
border of the ciliary process (O) and the iris border ( • ) relative to the epithelium
are also plotted.
and 45 within that part of the epithelium covered by the developing uvea.
From cell position 45 to the centre of the lens epithelium there is a slight and
gradual drop in the proportion of labelled cells. A somewhat similar distribution of labelled cells was previously recorded by Mikulicich & Young (1963)
in the lens of the newborn rat.
Cell elongation
(a) 6- to 7-week-old rats. Cell height was measured in sections and the results
are presented in Fig. 4. Flat cells comprising the central part of the lens epithelium are 4 /tm in height. Cuboidal cells in proliferation sub-compartment 1
range from 6 to 10 /tm in height. At the boundary of the proliferation compartment, cell position 17, there is a sharp increase in cell height towards the
lens equator.
(b) 1-day-old rats. Cell height was measured in sections of 1-day-old rat
lenses and recorded in Fig. 5. Cuboidal cells comprising most of the epithelium
are 10 /im in height. From the lens centre to the lens equator there is a gradual
increase in epithelial cell height to 25 /im at cell position 10. From cell position
10, as cell proliferation drops, there is a sharp increase in cell height towards
and beyond the equator.
Growth of the rat lens
157
y -Crystallm (-17)
§ -Crystallin (-5)
Elongation compartment
Proliferation compartment
•1140
120
30
100
9 25
[Extent of developing Border of
ciliary body
developing iris
20
80
I 15
60
10
40
=
U
20
•20
1
20
A
Lens equator
40
60
Cell position
80
100
120
140
•
Centre of epithelium
Fig. 5. Patterns of DNA synthesis (histogram) and cell elongation from lens
equator to the centre of the epithelium in 1-day-old rats. The extent of the developing ciliary body (O) and the border of the developing iris ( • ) relative to the
epithelium are also plotted.
Therefore the results from both 6- to 7-week-old and 1-day-old animals show
that the lens can be separated into two major compartments on the basis of cell
proliferation and cell height. From about the lens equator a proliferation
compartment extends anteriorly and an elongation compartment extends
posteriorly (see Fig. 1).
Distribution of crystallins in 1-day-old lenses
Alpha crystallin
Immunofluorescent studies with anti a-antibodies showed apple green
fluorescence throughout the cytoplasm of all lens cells (Fig. 6). No fluorescence
is found outside the lens showing that a-crystallin is specific to lens cells. All
epithelial and fibre cells fluoresce showing that a-crystallin is present in cells of
both proliferation and elongation compartments. This distribution of aII
EMB
44
158
J. W. McAVOY
Figs. 6-8. Typical distribution of crystallins in lens sections shown by immunofluorescence.
Fig. 6. a-Crystallin is detected in all lens epithelial (E) and fibre (F) cells and is not
detected outside the lens, e.g. in iris cells (I) or in cells of the ciliary body (CB).
Fig. 7. /?-Crystallin is first detected in elongating fibres about cell position ( — 5)
but is not detected in the epithelium.
Fig. 8. y-Crystallin is first detected in fibres about cell position (—17) but is not
detected in the epithelium.
crystallin in both the epithelial and fibre cells of the rat lens corresponds to that
in the bovine lens as shown by biochemical methods (Papaconstantinou, 1967)
and in the chick lens as shown by immunofluorescence (Ikeda & Zwaan, 1967;
Brahma & van Doorenmaalen, 1971).
Beta crystallin
Using anti /^-antibodies apple green fluorescence is not found in the cytoplasm
of all lens cells (Fig. 7). Beta-crystallin unlike a-crystallin is not detected in
epithelial cells (however, see below). Beta-crystallin is detected in the cytoplasm
of all fibre cells and in elongating cells from about cell position ( — 5) at the lens
equator. It is interesting that this is the position where cell division stops.
Thus, /?-crystallin is consistently detected only in cells of the elongation
compartment. This distribution in rat lens differs from that reported for /?crystallin in the chick (Zwaan, 1968) as shown by immunofluorescence and the
bovine lens (Papaconstantinou, 1967; van Kamp & Hoenders, 1973) as shown
by biochemical methods. While this difference may be due to a species difference
another explanation may be that antibodies used in the work reported here
may not cross-react with all the proteins generally thought to be within the
^-crystallin group and some of these may be found in the epithelium. In any
case, while the possibility that some /?-crystallin is localized in epithelial cells
Growth of the rat lens
159
of the rat lens cannot be completely excluded on the basis of the immunofluorescence results alone, these results show that from about cell position ( - 5 )
there are changes in the expression of /?-crystallin genes as cells elongate.
Gamma crystal/in
Using anti y-antibody apple green fluorescence is not found in the cytoplasm
of all lens cells (Fig. 8). Gamma is similar to /?-crystallin in that it is restricted
to fibre cells (however, see below) but different because it is detected later in the
elongation process, i.e. about position (—17) compared with about position
( - 5) for /?-crystallin.
It should be noted that in some sections a small number of epithelial cells
fluoresced after localization of y-crystallin (this also occurred occasionally after
localization of /?-crystallin but in these cases the fluorescence in epithelial cells
was very weak). However, in most cases the fluorescing cells were seen to be in
line with knife tears in the section. Therefore while it is difficult to exclude
completely the possibility that a small number of epithelial cells normally
contain y- and /?-crystallins, it seems much more likely that in some circumstances, e.g. knife tears in the section, these proteins move from the fibres into
some epithelial cells.
The distribution of y-crystallin in the rat lens as reported here appears to be
somewhat similar to that in amphibians (Takata, Albright & Yamada, 1966;
McDevitt, Meza & Yamada, 1969; McDevitt & Brahma, 1973; Brahma &
McDevitt, 1974) as shown by immunofluorescence and in the bovine lens as
shown by biochemical and immunofluorescence methods (Papaconstantinou^
1967). A different pattern of y-crystallin distribution in the 1-day-old rat lens
has been reported by Schubert, Trevithick & Hollenberg (1970). They localized
these proteins in cells throughout the epithelium, particularly in cells at the
lens equator. However, they found slight fluorescence in extralenticular sites
indicating diffusion of y-crystallin out of the lens fibres. Moreover, these
workers did not adequately test the specificity of their antibodies by immunodiffusion or immunoelectrophoresis. They did not show that their anti yantiserum did not react with a- and /?-crystallins. Consequently, the possibility
remains that their anti y-antibodies were contaminated by antibodies directed
against other lens proteins.
DISCUSSION
The results of this study show that the rat lens can be divided into two
functionally distinct compartments; a proliferation compartment and an
elongation compartment. These coincide with the anatomical separation of the
anterior and posterior parts of the lens by the ciliary body, and this separation
may be important in bringing about and maintaining functional differences
between compartments.
160
J. W. McAVOY
Proliferation compartment
The proliferation compartment is bathed by the aqueous humour. The
aqueous enters the anterior eye largely via the ciliary process, flows into the
posterior chamber (see Fig. 1) and then diffuses into the anterior chamber
where most of the outflow occurs (see Duke-Elder & Gloster, 1968 for review).
Since lens epithelial cells below the ciliary process in sub-compartment 1 divide
most often it is proposed that a proliferation factor(s) is a constituent of the
aqueous. The factor(s) may be diluted, inactivated or removed in the posterior
chamber and epithelial cells in this region, i.e. cells in sub-compartment 2,
would receive less stimulus to divide than cells directly below the ciliary process.
Furthermore, as the aqueous diffuses into the anterior chamber the concentration of the factor(s) may decrease further and thus epithelial cells in subcompartment 3 would receive a much reduced stimulus to divide. With this
model it is difficult to account for the slight but significant rise in mitotic
activity at the centre of the epithelium. However, these cells lie above the
sutures (see Fig. 1), are more tightly packed and have a more irregular shape
than the surrounding epithelial cells. It is possible therefore, that structural
differences may be related to their different proliferation properties.
The hypothesis that a proliferation factor(s) is present in the rat aqueous
derives support from lens injury experiments with frogs. It has been shown that
the injured frog eye contains a mitogenic factor that is transferable through the
aqueous (Weinsieder, Reddan & Wilson, 1976) and that all the major serum
proteins in the aqueous become elevated after injury (Weinsieder et ah 1975).
It seems possible, therefore, that levels of a proliferation factor(s) that is a
normal constituent of the aqueous become elevated as a result of injury. Also in
frogs it has been shown that after hypophysectomy there is no cell division in
the lens epithelium (van Buskirk, Worgul, Rothstein & Wainwright, 1975) even
after lens injury (Rothstein, van Buskirk, Gordon & Worgul, 1975). However,
when the proteins corresponding to growth hormone and prolactin are isolated
from the frog pituitary and administered individually, cell division is stimulated
in both intact and hypophysectomized animals (Wainwright, Rothstein &
Gordon, 1976). This work provides evidence, in frogs at least, that growth
hormone and/or prolactin may be involved either directly or indirectly in
control of cell division in the lens epithelium.
Elongation compartment
The elongation compartment in the posterior eye is bounded by the retina
and there is a large body of evidence that lens cell elongation and fibre cell
formation are normally dependent on the presence of this tissue. Coulombre &
Coulombre (1963) turned the lens of the 5-day-old chick through 180" so that
the epithelium which normally faced the cornea now faced the retina. Under
the influence of the new environment the epithelial cells elongated and gave
Growth of the rat lens
161
rise to fibre cells. This was found in similar experiments with mice eyes and it
was also shown that growth of the lens depended on the presence of the neural
retina (Yamamoto, 1976). In mice embryos it was shown that fibre cell elongation and fibre cell formation in culture were dependent on the presence of the
optic cup or neural retina and that this influence could pass through a Millipore
filter barrier of 25 /*m thickness and 0-45 /an. porosity (Muthukkaruppan, 1965).
Lens regeneration in newts, i.e. formation of new lens fibre cells from the iris
pigment epithelial cells after lens removal, is also dependent on the presence of
neural retina both in vivo (Stone & Steinitz, 1953; Stone, 1958; Reyer, 1971)
and in vitro (Yamada, Reese & McDevitt, 1973). Moreover, Yamada et al. have
shown that the regenerated fibre cells resemble normal fibre cells in that
y-crystallin is detected by immunofluorescence.
The factor(s) for lens induction and fibre cell formation in mice is thought
to be specific to the optic cup and neural retina (Muthukkaruppan, 1965). This
is based on the result that no lenses with fibre cells develop when presumptive
lens cells are cultured with spinal cord in place of the optic cup or neural retina.
However, in experiments with newts Jacobson (1958) showed that if the retinal
rudiment was removed at neurula stages 15 and 16, even before the lens placode
had formed, about 30 % of embryos developed with lens vesicles some with
early fibre formation. Just how much cell elongation takes place in those cases
is difficult to determine from the results presented, nevertheless they suggest
that cell elongation and fibre cell formation may not depend, in newts at least,
on a factor(s) specific to the developing retina. Further evidence that this may
be true for newts comes from lens regeneration experiments where the dorsal
iris epithelium transforms into lens with fibres when transplanted into a forelimb blastema containing regenerating nervous tissue (Reyer, Woolfitt &
Withersty, 1973) as well as when the regenerating nerves of the blastema are
transplanted into the anterior chamber next to the dorsal iris (Powell & Powers,
1973). Whole pituitary glands transplanted into the anterior chamber also
stimulate the transformation of iris epithelial cells into lens cells (Powell &
Segil, 1976). It has been concluded from these regeneration experiments with
newts that a 'neurotrophic substance' (Reyer et al. 1973) or a 'chemical
stimulus' (Powell & Segil, 1976) from nervous tissue is necessary for lens
regeneration from the iris. Chick lens epithelial cells will also elongate in
medium containing foetal calf serum in tissue culture (Philpott & Coulombre,
1965; Piatigorsky & Rothschild, 1972) and in cell culture (Okada, Eguchi &
Takeichi, 1971) and elongation in culture has been shown to resemble in vivo
elongation in that #-crystallin synthesis is stimulated in both cell (Okada et al
1971) and tissue (Milstone, Zelenka & Piatigorsky, 1976) culture. In the 6-day
chick lens epithelium it has been shown that insulin added to the culture
medium in place of serum will initiate elongation (Piatigorsky, 1973) and
stimulate ^-crystallin synthesis (Milstone & Piatigorsky, 1977). It has also been
shown in hyperplastic chick lenses that some cells in the inner layers of the
162
J. W. McAVOY
folded epithelium surrounded on all sides by epithelial cells, elongate into
short fibre-like cells (Clayton, 1975).
Thus, although cell elongation in vivo depends on a stimulus from the optic
cup or neural retina, in chicks and in some amphibians and perhaps in mammals,
the stimulus responsible can be given by other tissues, particularly nervous
tissues, by insulin and possibly by changes in cell-cell contact.
It is suggested, therefore, that a proliferation factor(s) that is a normal constituent of the aqueous controls cell division in the epithelium and an elongation
factor(s) that is normally produced or transmitted by the retina initiates the
formation of lens fibres. Thus, lens growth may depend on the presence of these
factors in their respective compartments of the eye. Furthermore, normal lens
growth may depend on a correct balance of the factors.
Distribution of crystallins
How the elongation factor(s) operates at the cellular and molecular levels is
of importance to the understanding of lens cell specialization. In this study it
was shown that in the 1-day-old rat lens cell elongation begins about position
10, cell division stops at position ( — 5), this is about where /?-crystallin is first
detected and finally y-crystailin is localized at about position (—17). Therefore,
elongation begins before ft- and y-crystallins are detected. A somewhat analogous situation has been demonstrated in the chick. Lens epithelial cells from
6-day chicks in culture doubled in length in the absence of new protein synthesis
(Piatigorsky, Webster & Wollberg, 1972). It was shown that the initial doubling
in length of epithelial cells from 6-day-old chick lenses occurred even if protein
synthesis was inhibited by cycloheximide. However, further elongation depended
upon new protein synthesis. The initial elongation was inhibited by colchicine
which prevents the organization of microtubules. It was concluded from this
work that elongation in chick epithelial cells depends, firstly, on the organization of pre-existing microtubule elements and, secondly, on changes in patterns
of protein synthesis. Elongation in the rat lens in vivo may also be separated
into these two stages since elongation begins before new proteins are detected.
Beta- and y-crystallins are different from a-crystallin in that they are only
localized in cells of the elongation compartment. Thus, synthesis of /?- and
y-crystallins may normally depend on the elongation factor(s) from the retina.
Also, since there is a close relationship between cessation of cell division and
detection of /?-crystallin, another factor governing the synthesis of /?-crystallin
may be whether the cells have ceased dividing. In this way the expression of
crystallin genes may be co-ordinated as the lens grows and cells make the
transition from proliferation to elongation compartments.
Growth of the rat lens
163
I am grateful to Dr J. J. Harding for his advice and assistance on the separation of the
lens proteins. Thanks also go to Ms B. Galdes and Mr A. Turner for their skilled technical
assistance and to Ruth van Heyningen, Ruth Clayton, Marie Dziadek and J. J. Harding for
their helpful comments on the manuscript. The support for this research was provided by
grant No. t.ROI.EY01548-01 awarded by the N.E.I, to Dr Ruth van Heyningen to whom
1 also owe special thanks for her continued interest in this work.
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{Received 12 August 1977, revised 26 October 1977)