Cell division, cell elongation and distribution of a-, (3- and y-crystallins in the rat lens

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1 /. Embryo/, exp. Morph. Vol. 44, pp , Printed in Great Britain Company of Biologists Limited 1978 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 6- to 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.

2 150 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 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.

3 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 has an average mitotic index of 0-27%. Sub-compartment 2 (SC 2) from about cell position has an average mitotic index of 009% and sub-compartment 3 (SC 3) from about cell positions 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 [ 3 H]- thymidine autoradiography. Six rats were each given an intra-peritoneal injection of 10 /tci/g body weight of methyl-[ 3 H]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

4 152 J. W. McAVOY '% o >-> 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 = /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

5 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 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 a- or /?-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 y- crystallins, 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

6 154 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

7 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 [ 3 H]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

8 156 J. W. McAVOY Cell position 17 Elongation compartment Anterior border of ciliary process 01-1 ^ - ^ ^ -5 Lens equator Cell position 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.

9 Growth of the rat lens 157 y -Crystallm (-17) -Crystallin (-5) Elongation compartment Proliferation compartment [Extent of developing Border of ciliary body developing iris I = U A Lens equator Cell position 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 a- II EMB 44

10 158 J. W. McAVOY Figs 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

11 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 y- antiserum 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.

12 160 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

13 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

14 162 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.

15 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.ey 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. REFERENCES BRAHMA, S. K. & VAN DOORENMAALEN, W. J. (1971). Immunofiuorescence studies of chick lens F1SC and a-crystallin antigens during lens morphogenesis and development. Ophthal. Res. 2, BRAHMA, S. K. & MCDEVITT, D. S. (1974). Ontogeny and localization of gamma-crystal 1 ins in Rana temporaria, Ambystoma mexicanum and Pleurodeles waltlii normal lens development. Expl Eye Res. 19, CLAUSEN, J. (1969). Immunochemical techniques for the identification and estimation of macromolecules. Laboratory Techniques in Biochemistry and Molecular Biology, vol. 1 (ed. T. S. Work & E. Work). Amsterdam: North Holland Publishing Co. CLAYTON, R. M. (1974). Comparative aspects of lens proteins. The Eye, vol. 5 (ed. H. Davson & L. T. Graham), pp London: Academic Press. CLAYTON, R. M. (1975). Failure of growth regulation of the lens epithelium in strains of fast-growing chicks. Genet. Res. 25, COULOMBRE, J. L. & COULOMBRE, A. J. (1963). Lens development: fibre elongation and lens orientation. Science, N.Y. 142, DUKE-ELDER, W. S. & GLOSTER, J. (1968). The aqueous humour. System of Ophthalmology, vol. 4 (ed. W. S. Duke-Elder), pp London: Henry Kimpton. HANNA, C. & O'BRIEN, J. E. (1961). Cell production and migration in the epithelial layer of the lens. Archs Ophthal. 66, HARDING, J. J. & DILLEY, K. J. (1976). Structural proteins of the mammalian lens: a review with emphasis on changes in development, ageing and cataract. Expl Eye Res. 22, HARDING, C. V., HUGHES, W. L., BOND, V. P. & SCHORK, P. (1960). Autoradiographic localization of tritiated thymidine in whole-mount preparations of lens epithelium. Archs Ophthal. 63, HARDING, C. V., REDDAN, J. R., UNAKAR, N. J. & BAGCHI, M. (1971). The control of cell division in the ocular lens. Int. Rev. Cytol. 31, HOWARD, A. (1952). Whole mounts of rabbit lens epithelium for cytological study. Stain Technol.ll, IKEDA, A. & ZWAAN, J. (1967). The changing cellular localization of a-crystallin in the lens of the chicken embryo, studied by immunofiuorescence. Devi Biol. 15, JACOBSON, A. G. (1958). The roles of neural and non-neural tissues in lens induction. /. exp. Zool. 139, MCAVOY, J. W. & DIXON, K. E. (1978). Cell proliferation and renewal in the small intestinal epithelium of adult Xenopus laevis. J. exp. Zool. (In the Press.) MCDEVITT, D. S. & BRAHMA, S. K. (1973). Ontogeny and localization of the crystallins during embryonic lens development in Xenopus laevis. J. exp. Zool. 186, MCDEVITT, D. S., MEZA, I. & YAMADA, T. (1969). Immunofiuorescence localization of the crystallins in amphibian lens development, with special reference to the y-crystallins. Devi Biol. 19, MIKULICICH, A. G. & YOUNG, R. W. (1963). Cell proliferation and displacement in the lens epithelium of young rats injected with tritiated thymidine. Invest. Ophthalmol. 2, MILSTONE, L. M. & PIATIGORSKY, J. (1977). #-crystallin gene expression in embryonic chick lens epithelia cultured in the presence of insulin. Expl Cell Res. 105, MILSTONE, L. M., ZELENKA, P. & PIATIGORSKY, J. (1976). 5-crystallin mrna in chick lens cells; mrna accumulates during differential stimulation of 5-crystallin synthesis in cultured cells. Devi Biol. 48,

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