Exp. Eye Res. (1992) 54, 193-200
Lens Fiber Cell Differentiation and Expression of Crystallins in Co-cultures of Human Fetal Lens Epithelial Cells and Fibroblasts CHANDRASEKHARAM
N. N A G I N E N I *
AND S U R A J P. BHAT'~
Jules Stein Eye Institute, University of California, School of Medicine, Los Angeles, CA 90024-1771, U,S.A. (Received Bethesda 5 February 1991 and accepted in revised form 5 March 1991) Growth of the ocular lens is directed by the division and differentiation of a single layer of epithelial cells located at the equatorial region. It is conceivable that this region of the lens capsule presents a special microenvironment modulated by molecular cues emanating from the surrounding tissues. In an effort to investigate the source and nature of these molecular cues, we co-cultured human fetal lens epithelial cells and fibroblasts derived from the ciliary body. We observed morphological differentiation as evidenced by the appearance of differentiating lentoid structures associated with fibroblasts. Characterization of the expression of lens-specific proteins revealed that in addition to ~B-crystalfin, these lentoid structures contain the lens fiber cell-specific proteins, aA-crystallin, flB~-crystallin and yS-crystafiin. None of these crystallins could be found in the surrounding undifferentiated lens epithelial cells. Interestingly, aBcrystallin usually present in lens epithelial cells when cultured alone, was found to be markedly decreased, both in synthesis and content in the cells surrounding the differentiated structures, suggesting that the process of differentiation in vitro may concomitantly produce a factor(s) which modulates aBcrystallin expression in these cells. Key words: lens; epithelium; differentiation; crystallins; co-culture; fibroblasts.
1. Introduction The development of the ocular lens presents an attractive paradigm for the study of the process of differentiation and its attendant molecular components. Based on embryological investigations it has been suggested that polarity of differentiating lens fiber cells at the equatorial zone is determined by extrinsic factors (Coulombre and Coulombre, 1963). Historically, these factors have been considered to be derived from the neuroretina. This consideration has been based on the proximity of the optic vesicle to the presumptive lens ectoderm during the early phase of lens induction and the observation of differentiating posterior cells of the lens vesicle. However, in the postembryonic lens the differentiation of lens epithelial cells is spatially restricted to the equatorial region (Maisel et al., 1981) which is next to the surrounding ciliary body. It is obvious from the spatially restricted, differentiation zone that the equatorial region presents a different microenvironment than the rest of the capsule surrounding the lens. It has been assumed by a number of investigators that the special environment in this differentiating zone is modulated by molecular cues coming from the retina, diffusing through the vitreous and concentrating at the equatorial regions. This rationale has guided investigators to examine the response of lens epithelial cells to neuroretina or the * Present address: Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD 20862, U.S.A. ~" For correspondence.
00144835/92/020193 +08 $03.00/0 13
extracts~factors derived from the neuroretina. These responses have varied from proliferation of the epithelial cells to the promotion of differentiation (McAvoy, 1980; Arruti, Ciriflo and Courtois, 1985; Courty et al., 1986; Piatigorsky and Zelenka, 1990, for review). One of the first factors shown to promote differentiation in epithelial cell explants was isolated from chicken vitreous and characterized to be Somatomedin C or insulin-like growth factor (Beebe et al., 1987). Explants from the rat lens epithelium have been shown to respond to fibroblast growth factor by differentiation (Chamberlain and McAvoy, 1987). Other factors such as Somatomedin C (Rothstein et al., 1980) and PDGF (Brewitt and Clark, 1988) have been suggested to be involved in epithelial cell proliferation and lens growth, respectively. Thus, at the present time the source(s) and the type of stimuli that modulate lens differentiation remain conjectural. In this report we present investigations conducted on lens fiber cell differentiation in vitro from the perspective of cellular interactions. This approach is based on epithelial-mesenchymal interactions known to induce differentiation and specific gene expression during early development (Grobstein, 1954; Kratowil and Schwartz, 1976; Jacobson and Sater, 1988; Sanders and Bouziges, 1988; Reichmann et al., 1989). We have co-cultured h u m a n lens epithelial cells with fibroblasts. The source of fibroblasts was selected on the following basis: the equatorial region is in close juxtaposition to the ciliary body (Nishida, 1982) which has been invoked to influence lens growth within this area (Mikuficich and Young, © 1992 Academic Press Limited EER 54
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FIG. 1. Phase contrast photomicrographs of cultures of human fetal lens epithelial cells (LEC) and human fetal ciliary body fibroblasts (CBF). A, Pure population of confluent CBF. B, CBF plated at a lower density to form non-confluent cultures producing a network of connections between the ceils. C, Secondary culture of a pure population of confluent LEC. D, LEC and CBF after 4 weeks of co-culture showing the characteristic fiber network (arrows) produced by the fibroblasts. Bar = 200 #m. 1963 ; Young and Ocumpaugh, 1966). As a first step we cultured fibroblasts from the surrounding tissue (ciliary body) and then investigated the response of h u m a n fetal lens epithelial cells (Nagineni and Bhat, 1988) to co-culture with these cells. The response which manifests morphologically in differentiating fibers (lentoid bodies, Okada, Eguchi and Takeichi, 1971) was characterized by following the appearance a n d / o r disappearance of lens fiber cell-specific proteins.
2. Materials and Methods
Cell Cultures Human fetal lens epithelial cells (LEC) and pure populations of h u m a n fetal ciliary body fibroblasts (CBF) derived from h u m a n fetuses of 1 8 - 2 4 weeks of age, were cultured from the lens capsule-epithelium and ciliary body explants, respectively, as described earlier (Nagineni and Bhat, 1988, 1989a, b). Pure populations of LEC and CBF in primary culture were dissociated with trypsin-EDTA and seeded at a 9:1 (LEC:CBF) ratio for co-culture.
Analysis of Fibroblast-associated Lentoid Structures The fiber network and associated lentoid structures produced in the co-cultures of LEC and CBF were
removed easily by holding with fine forceps at one end and lifting it away. The entire fiber network was interconnected and could be peeled away leaving the undifferentiated lens epithelial cells behind. These structures were collected from five to eight 35-mm culture dishes and pooled for analysis. Supernatant fractions of human fetal lens, LEC and CBF were prepared as described earlier (Nagineni and Bhat, 1988, 1989a, b). Cell cultures or fiber network and its associated cells were extracted with lysis buffer [10 mM Tris (pH 7"4), 100 mM NaC1, 2 mM EDTA 1% NP-40, 0'5% deoxycholate and 0.5% triton X-lO0]. After freezing and thawing four times on dry ice, the lysate was centrifuged at 1 7 0 0 0 g for 20 min (Sorvall, RC5C, Dupont, Wilmington, DE) and the supernatant used for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) a n d / o r immunoblotring (Nagineni and Bhat, 1988).
Immunoblotting Proteins in the supernatant fractions were separated on 15% SDS-PAGE minigels (Hoeffer Inc., San Francisco, CA). After transferring proteins onto nitrocellulose (Schleicher and Schuell, Inc., Keene, NH), the membranes were incubated in the presence of specific rabbit-antisera to different crystallins. HRP (Horse radish peroxidase) conjugated goat anti-rabbit IgG was used as the second antibody and colour was
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developed with H202 and p-chloro 1-napthol (Biorad Laboratories, Sacramento, CA).
Labeling of Cells Fibroblast-associated lentoid structures (including the fiber network) were removed 2 days before labeling to prevent contamination of undifferentiated parts of culture dish. Labeling with pS]methionine (trans asSlabel, ICN Radiochemicals, Irvine, CA), preparation of supernatant fractions ( 1 7 0 0 0 g, 20 min) and SDSPAGE in 159/o gels were done as described above. 3. Results
Figure 1 shows the morphology of h u m a n fetal lens epithelial cell cultures and epithelial:fibroblast co-
Fro. 2: LEC co-cultured with CBF. A and B, After 10-12 weeks of co-culture showing translucent lentoid structures (*). C, Lentoid bodies (arrows) at the termini of the fiber network produced by fibroblasts. Bar = 100 #m.
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cultures. Co-cultures of LEC and CBF resulted in the appearance of a large number of lentoid bodies. Lentoid bodies (Okada et al., 1971) are the morphological manifestation of differentiation of lens epithelial cells into lens fiber cells. These in vitro differentiated structures have previously been shown to contain elongated differentiated fiber ceils and have been characterized by immunofluorescence and at the electron microscopic level (Okada et al., 1971; Hamada and Okada, 1978; Arita et al., 1990). Interestingly, in our system these translucent, differentiating structures were seen predominantly associated with fibroblasts which formed a fiber network surrounded by undifferentiated epithelial cells [Fig. I(D)]. Such structures are rarely observed when lens epithelial cells are cultured alone (Nagineni and Bhat, 1988), as shown in Fig. I(C). The fibroblasts used in this study, grown at high density, are shown in Fig. I(A), However, when these cells were grown at a lower density, they grew as a network of isolated cell masses in contact with each other [Fig. I(B)]. The results of co-culture of lens epithelial cells such as the ones shown in Fig. I(C) and fibroblasts [Figs I(A) and (B)] in a 9 : 1 ratio are presented in Fig. 1 (D) and Figs 2(A), (B) and (C). The lopsided ratio in favour of lens epithelial ceils had to be maintained in order to prevent inherently faster growing fibroblasts from taking over the entire culture dish. This ratio also helped in defining the boundaries of fibroblast growth within a monolayer of lens epithelial cells. Comparison of Fig. 1 (B) with Fig. I(D) suggests that the network in co-cultures (indicated by arrows) appears to be contributed by fibroblasts independent of epithelial cells. Figure I(D) is a 2-3-week-old co-culture in which transparent lentoid structures have just started to appear. Figures 2(A), (B) and (C) represent 10-12week-old co-cultures. Lentoid structures in different areas are seen associated with the fiber network formed by fibroblasts. The translucent, differentiated structures, which we presume are the differentiating fiber ceils derived from the lens epithelial cells, are found associated with this network [Figs 2(A), (B) and (C)]. Interestingly, these translucent lens fiber cells are also seen at the termini of these fibrous structures [Fig. 2(C)]. All of these structures show positive reactivity with 7-crystalfin antiserum as detected by immunofluorescence (data not shown). A thorough biochemical analysis of the differentiated structures associated with the fibroblasts was undertaken using antibodies specific to four different crystallins (aA, aB, fiB2, yS), representing three major immunologically distinct classes of proteins (a, fl and 7-crystallins) (Wistow and Piatigorsky, 1988 ; Bloemendal, Piatigorsky and Spector, 1989). These proteins are known to be present in terminally differentiated fiber cells (Bloemendal, 1981 ; Wistow and Piatigorsky, 1988). Alpha A-crystallin (aA) and aB-crystallin (aB) are assumed to be two subunits of the main structural protein a13-2
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C,N. N A G I N E N I AND S.P. B H A T 4
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FIG. 3. Demonstration of lens fiber cell-specifc expression in lentoid structures associated with fibroblast generated network. The fiber network produced by CBF and its associated lentoid structures was peeled away with forceps and analysed by immunoblotting. A, Immunoblot with anti-a-crystallin (all the lanes are from the same blot, processed identically). Each lane designation is followed by the amount of protein loaded, in parentheses, le, Lens homogenate (5 #g). Lane 1, Fiber network collected from secondary cultures of LEC [5 weeks PC (primary culture), 24 weeks SC (secondary culture) co-cultured with CBF (45 #g)]. Lane 2, Fiber network collected from secondary cultures of LEC (12 weeks PC, 33 weeks SC), co-cultured with CBF (45 #g). Lane 3, CBF (80 #g). B, Immunoblot analysis with anti-a and anti-fiB 2 (this blot was first used with anti-a and then re-used with anti-fiBs; all the lanes are from the same blot). Lanes 1, 2 and 3 as in (A). Lane 4, Tertiary cultures (TC) of LEC that had about 80% of refractile elongated cells (Nagineni and Bhat, 1989a) (2 weeks PC, 3 weeks SC, 1 week TC: 12 #g). C, Immunoblot analysis with anti- 7. Lane 1, Secondary cultures of LEC (8 weeks PC, 25 weeks SC: 10/zg). Lane 2, Tertiary cultures of LEC (8 weeks PC, 25 weeks SC, 25 weeks TC: 10 #g). Lane 3, Pellet fraction of the cells in lane 2 (16 #g). Lane 4, Fiber network collected from secondary cultures of LEC co-cultured with CBF [same as in lane l in (A) and (B)] (20 #g). Lane 5, fiber network collected from secondary cultures of LEC co-cultured with CBF [same as lane 2 in (A) and (B) : 20 #g). Lane 6, CBF (80 #g). [In (C) the first lane contains molecular mass standards, same as shown in Fig. 4. Standards are not shown in (A) and (B).]
crystallin present in the lens fiber cells (see Discussion). BetaB2-crystallin (fiB2) is synthesized upon differentiation of lens epithelial cells into fibers, and 7-crystallins (7) are known to be synthesized during early growth of the lens, specifically in the fiber cells. Figure 3 shows that at least one representative of each of these classes of crystallins can be detected within the fibroblastassociated network which contains the translucent lentoid structures [Fig. 3(A) lanes 1 and 2 (aA and ~B), Fig. 3(B) lanes 1 and 2 (fiB2), and Fig. 3(C) lanes 4 and 5 (7S)]. The data presented in Fig. 3 was obtained from two different sets of cultures (lanes 1 and 2). This may explain the variations in the content of each protein detected on the immunoblots [e.g. lanes 1 and 2, in Figs 3(A) and (B) and lanes 4 and 5 in Fig. 3(C), represent two different sets of cultures]. A blot, similar to that presented in Fig. 3(A) was first used for aA immunoblotting and then re-used for analysis with
anti-fiB 2 [Fig. 3(B) lanes ] and 2]. It must be stressed that all these proteins have been well characterized and their positions in an SDS-PAGE well recognized (Bloemendal, 1981). Also, the antisera used in the experiments shown in Fig. 3 have been well characterized earlier (Nagineni and Bhat, 1988, 1989a, b; McFall-Ngai et al., 1985, 1986). The anti-~-crystallin antiserum reacts with both ~A and aB. Anti-flB~ specifically reacts with flB~ and anti- 7 reacts with different members of 7-crystallin family of proteins, all of which fall in the molecular mass range of 2 0 - 2 4 kDa. Among these proteins yS moves as a component of about 24 kDa molecular mass. Fibroblast extracts do not show reactivity with any of the antisera used here [Figs 3(A) and (B) lane 3 and Fig. 3(C) lane 6]. The data in Fig. 3 indicate that all classes of crystallins (a, fiB~ and 7S) could be detected only in the differentiating structures. It was of interest to
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45-
compares them to the cells which have never been cocultured with fibroblasts (compare lanes 1, 2 and 7 with lanes 3, 5 and 6). Inhibition of the synthesis of ~B in undifferentiated epithelial cells stands out clearly. Furthermore, by immunoblotting with an ~B-specific antibody [Figs 4(A) and (B) lanes 3, 5 and 6], an appreciable decrease is seen in the content of this protein.
30-
4. Discussion
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FIG. 4. Inhibition of the expression of aB in undifferentiated LEC surrounding the differentiated structures. A, Autoradiogram of ~SS-proteins of epithelial cells labeled after removal of the fibroblast associated lentoid structures. Lane 1, Primary Cultures of pure population of LEC (total age in culture, 13 weeks). Lane 2, Secondary cultures (5 weeks PC, 13 weeks SC) of pure population of LEC. Lane 3, Secondary cultures of LEC (5 weeks PC, 13 weeks SC), co-cultured with CBF. Lane 4, Tertiary cultures of pure population of LEC (5 weeks PC, 4 weeks SC, 10 weeks TC). Lane 5, Secondary cultures of LEC (5 weeks PC, 13 weeks SC) co-cultured with CBF. Lane 6, Secondary cultures (5 weeks PC, 17 weeks SC) of LEC co-cultured with CBF. Lane 7, Tertiary cultures of pure population of LEC (5 weeks PC, 4 weeks SC, 15 weeks TC). Numbers on the left side of panel indicate the positions of standards. B, Immunob!ot analysis with anti-aB antibody: le, human fetal lens extract (2 #g) run on the two outside lanes of the immunoblot. The rest of the lanes correspond to lanes 1-7 in (A). Numbers on the left side of the immunoblot show positions of prestained protein markers.
ascertain the synthesis of proteins in the undifferentiated epithelial cells surrounding the fibroblast associated network. An immunoblot with anti-aA, anti-fiB 2 or anti- 7 of the proteins extracted from the undifferentiated cells did not show any reactivity (data not shown). Figure 4(A) shows an SDS-PAGE autoradiograph of proteins synthesized in these cells and
Growth and differentiation of the ocular lens has been primarily studied in the context of the effect of molecular influences supposedly originating from the neuroretina or the back of the eye. These studies have been based on the rationale that lens morphogenesis is induced by the emerging optic vesicle and that this molecular influence/interaction continues into the post-embryonic growth of the lens (McAvoy, 1980). Although the role of optic vesicle/retina in lens induction is questionable (Karkinen-Jaaskelainen, 1978: see Jacobson and Sater, 1988), it is possible that the retina exerts some influence on the adult lens (Yamamoto, 1976). A developmental and molecular basis for differentiation of lens epithelial cells into f b e r cells during early embryogenesis and in the postembryonic lens remains poorly understood. The spatial restriction of the differentiating lens fiber cells lends itself to speculation that the microenvironment in the differentiating zone m a y be modulated by molecular cues coming laterally from a tissue in the immediate vicinity of the equatorial zone. It must, however, be recognized that these two possible trophic influences, namely the neuroretina and the ciliary body, need not be mutually exclusive. It should also be noted that differentiation in cultured epithelial cells has been shown to be dependent on the culture dish substrate (Arita et al., 1990) and it has been demonstrated that the lens capsule (basement m e m b r a n e components) has a role in promoting differentiation of lentoid bodies in vitro (Muggleton-Harris and Higbee, 1987). Experiments presented here represent our initial attempts at understanding the special microenvironment offered by the equatorial region in the lens. The data presented above demonstrate that co-culture of lens epithelial cells and fibroblasts results in the appearance of lentoid bodies at the interface of the epithelial-flbroblast contact. Whether there is indeed communication between the lens and the surrounding tissues, particularly in the area of the equatorial zone and whether it is only the ciliary body fibroblasts that promote differentiation remains to be investigated. A further unknown is the embryological origin of the fibroblasts. The results, however, present a basis for systematic exploration of the effect of tissues surrounding the differentiating zone of the adult lens. The fact that translucent structures containing lensspecific proteins are generated upon co-culture of the
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fetal lens epithelial with fibroblasts suggests that the fibroblasts are either providing a modified extracellular matrix, or a t r o p h i c factor (Grobstein, 1954; Beebe et al., 1987; Muggleton-Harris and Higbee, 1987; Jacobson and Sater, 1988; Sanders and Bouziges, 1_988; Reichmann et al., 1989) or that they physically interact with epithelial cells through surface molecules (for example Anklesaria et al., 1990; Fehon et al., 1990). We assume that the lentoid bodies are derived from the fetal lens epithelial i n this co-culture. Independent subculturing of fibroblasts over long periods of time did not show any appearance of lentoids. Alternatively, it is possible that the lentoid bodies are derived via transdifferentiation of fibroblasts and that this event is triggered by co-culture with epithelial cells. Transdifferentiation, however, is a spontaneous phenomenon associated with pleuripotent cells. Although unlikely, from an embryological perspective, the data presented above do not allow negation of this possibility. Further experiments with molecularly tagged cells will be needed to establish the source of lentoid bodies with certainty. Interesting aspects of the expression of crystalfns deserve mention. We have previously shown that the appearance of the fiber cell-specific protein flB~ was dependent on subculturing and not associated with any morphological manifestation such as the appearance of lentoid bodies (Nagineni and Bhat, 1988, 1989a). The expression of fiB 2 [Fig. 3(B) lanes 1 and 2] indicates that this protein may be activated by at least two alternative pathways, one of which is associated with morphological differentiation. It is important to point out that 7S increases postnatally in fiber cells in h u m a n lens (Thomson and Augusteyn, 1985). Other 7-crystallins are predominantly synthesized during fetal life and may represent predominant contributions of the primary fiber cells which are derived from the posterior cells of the lens vesicle during lens morphogenesis. Gamma S therefore represents a 7-crystallin synthesized in secondary fiber cells which are derived from the anterior epithelial cells. There are no posterior epithelial cells available in the lens vesicle after the sixth week of gestation (Moore, 1982), all of them having been transformed into primary fibers which are restricted to the fetal nucleus of the lens. Therefore, the observation [Fig. 3(C), lanes 4 and 5] that 7S, of all the 7-crystallins, is expressed upon differentiation in vitro by lens epithelial cells may represent a true in vivo expression potential of these cells with respect to the synthesis of the 7-crystallin group of proteins. The differentiated structures produced in the cocultures along with the fiber network can be removed leaving behind undifferentiated epithelial cells. This may be due to the differential expression of cell adhesion molecules (Takeichi, 1988). Analysis of the proteins synthesized by the undifferentiated cells revealed an almost specific inhibition of ~B synthesis in these cells. This observation is novel in that it is only
C.N. N A G I N E N I A N D S.P. B H A T
associated with the undifferentiated cells and not the differentiated ones. This is intriguing considering that lens epithelial cells, when cultured alone, always synthesize c~B [Nagineni and Bhat, 1988, 1989a, b; Fig. 3(B) lane 4, and Fig. 4 lanes 1, 2, 4 and 7]. This observation must be examined in the light of recent findings demonstrating aB to be an extra-lenticular protein present in a number of other tissues (Bhat and Nagineni, 1989; Dublin et al., 1989; Iwaki et al., 1989) suggesting that it may have other noncrystallin functions, Based on its presence in epithelial cells (Nagineni and Bhat, 1989b) and its appearance on a temporal basis in different tissues (Bhat and Nagineni, 1989) and the recent demonstration of the accumulation of this protein in 3T3 cells expressing v-mos and Ha-ras oncogenes (Klemenz et al., 1991) it may be hypothesized that this protein could have a role in differentiation of a variety of tissues. From the data presented above it is not possible to causally link differentiation and the inhibition of ~B in the undifferentiated cells. However, the fact that ~B continues to be synthesized in lens epithelial cells cultured alone indicates that either fibroblasts or the process of differentiation per se produces factor(s) which inhibit the synthesis of this protein in the cells surrounding the differentiating lens fbers. The data suggest a diffusible modulator of ~B synthesis. It is tempting to speculate that the inhibition of c~B in the adjacent cells may represent a molecular link in a scheme of events that brings about the inhibition of differentiation in the neighboring cells, a mechanism akin to that proposed for neuro-genesis in Drosophila, wherein a differentiating neuroblast within a sheet of neurogenic cells inhibits the adjacent cells from differentiating (Doe and Goodman, 1985). We believe that the differentiation at the interface of the epithelial-fibroblast cells reported here represents a paradigm for the study of the microenvironment in the equatorial region of the postembryonic lens. Clearly, further investigations are warranted to delineate the molecular nature of these interactions and whether eB has any direct role in any of these processes or is merely an associated indicator.
Acknowledgements We thank Dr Joseph Horowitz for providing c~- and fiB2crystallin antisera, Dr Bill O'Day for the human fetal lenses and Dr Dean Bok for critical reading of the manuscript. This work was supported by National Eye Institute, National Institutes of Health, Bethesda, MD (grant no. EY06044, S.P.B.). S.P.B. is a Research to Prevent Blindness William and Mary Greve International Scholar.
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forming growth factor a to epidermal growth factor receptors promotes cell proliferation. Proc. Natl. Acad. Sci. U.S.A. 87, 3289-93. Arita, T., Lin, L., Susan, S.R. and Reddy, V.N. (1990). Enhancement of differentiation of human lens epithelial in tissue culture by changes in cell-substrate adhesion. Invest. Ophthalmol. Vis. Sei. 31, 2395-404. Arruti, C., Cirillo, A. and Courtois, Y. (1985). An eye-derived growth factor regulates epithelial cell proliferation in the cultured lens. Differentiation 28, 286-90. Beebe, D.C., Silver, M.H., Belcher, K.S., VanWyk, J.J., Svoboda, M. E. and Zelenka, P. S. (1987). Lentropin, a protein that controls lens fiber differentiation, is related functionally and immunologically to the insulin-like growth factors. Proc. Natl. Acad. Sci. U.S.A. 84, 2327-30. Bhat, S. P. and Nagineni, C. N. (1989). aB subunit of lensspecifc protein ~-crystafiin is present in other ocular and non-ocular tissues. Biochem. Biophys. Res. Commun. 158, 319-25. Bloemendal, H. (1981). Molecular and Cellular Biology of the Eye Lens. John Wiley and Sons: New York. Bloemendal, H., Piatigorsky, J. and Spector, A. (1989). Recommendations for crystallin nomenclature. Exp. Eye Res. 48, 465-6. Brewitt, B. and Clark, J. I. (1988). Growth and transparency in the lens, an epithelial tissue, stimulated by pulses of PDGF. Science 242, 777-9. Chamberlain, C.G. and McAvoy, J.W. (1987), Evidence that fibroblast growth factor promotes lens fiber differentiation. Curr. Eye Res. 6, 1165-8. Coulombre, ]. L. and Coulombre, A.J. (1963). Lens development : fiber elongation and lens orientation. Science 142, 1489-90. Courty, ]., Chevallier, B., Moenner, M., Loret, C., Lagente, O., Bohlen, P., Courtois, Y. and Barritault, D. (1986). Evidence for FGF-like growth factor in adult bovine retina: analogies with EDGF I. Biochem. Biophys. Res. Commun. 136, 102-8. Doe, C.Q. and Goodman, C.S. (1985). Neuro-genesis in grasshopper and fushi-tarazu Drosophila embryos. Cold Spring Harbor Syrup. Ouant. Biol., L, 891-903. Dubin, R. A., Wawrousek, E. F. and Piatigorsky, J. (1989). Expression of murine ~B-crystallin gene is not restricted to the lens. ]. Mol. Cell Biol. 9, 1083-91. Fehon, R. G., Kooh, P. ]., Rebay, I., Regan, C.L., Xu, T., Muskavitch, M.A.T. and Artavanis-Tsakonas, S. (1990). Molecular interactions between the protein products of the neurogenic loci notch and delta, two EGF-homologous genes in Drosophila. Cell 61, 523-34. Grobstein, C. (1954). Tissue interactions in the morphogenesis of mouse embryonic rudiments in vitro. In Aspects of Synthesis and Order in Growth (Ed, Rudnick, D.). Pp. 233-56. Princeton University Press: Princeton, New Jersey. Hamada, Y. and Okada, T. S. (1978). In vitro differentiation of cells of the lens epithelial of human fetus. Exp. Eye Res. 26, 91-7. Iwaki, T., Kume-Iwaki, A., Liem, R. K. H. and Goldman, J. E. (1989). ~zB-crystallin is expressed in non-lenticular tissues and accumulates in Alexander's disease brain. Cell 57, 71-8. Jacobson, A.G. and Sater, A.K. (1988). Features of embryonic induction. Development 104, 341-59. Karkinen-]aaskelainen, M. (1978). Permissive and directive interactions in lens induction. ]. Embryol. Exp. Morphol. 44, 167-79. Klemenz, R., Frohli, E., Aoyama, A., Hoffmann, S., Simpson, R. J., Moritz, R. L. and Schafer, R. (1991). aB-crystallin accumulation is a specific response to the Ha-ras and v-
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mos oncogene expression in transformed mouse NIH 3T3 fibroblasts. Mol. Cell Biol. 11, 803-12. Kratowil, K. and Schwartz, P. (1976). Tissue interaction in androgen response of embryonic mammary rudiment of mouse. Identification of target tissue for testosterone. Proc. Natl. Acad. Sci. U.S.A. 73, 4041-4. Maisel, H., Harding, C.V., Alcala, T.R., Kuszak, T. and Bradley, R. (1981). The morphology of the lens. In Molecular and Cellular Biology of the Eye Lens. (Ed. Bloemendal, H.). Pp. 49-84. John Wiley and Sons: New York. McAvoy, J. W. (1980). fl and 7-crystallin synthesis in rat lens epithelial explanted with neural retina. Differentiation 17, 85-91. McFafi-Ngai, M. J., Ding, L. L., Takemoto, L. ]. and Horowitz, J. (1985). Spatial and temporal mapping of the agerelated changes in human lens crystallin. Exp. Eye Res. 41, 745-58. McFall-Ngai, M., Horowitz, Ding, L. and Lacey, L. (1986). Age-dependent changes in the heat-stable crystallin, flBp of the human lens. Curr. Eye Res. 5, 387-94. Mikulicich, A. G. and Young, R. W. (1963). Cell proliferation and displacement in the lens epithelium of young rats injected with tritiated thymidine. Invest. Ophthalmol. 2, 344-54. Moore, K. L. (1982). The Developing Human. Pp. 413-423. W. B. Saunders Co. : London. Muggleton-Harris, A.L. and Higbee, N. (1987). Factors modulating mouse lens epithelial cell morphology with differentiation and development of a lentoid structure in vitro. Development 99, 25-32. Nagineni, C. N. and Bhat, S. P. (1988). Maintenance of the synthesis of c~B-crystallin and progressive expression of flBp-crystallin in human fetal lens epithelial cells in culture. Dev. Biol. 130, 402-5. Nagineni, C. N. and Bhat, S. P. (1989a). Human fetal lens epithelial cells in culture : an in vitro model for the study of crystafiin expression and lens differentiation. Curr. Eye Res. 8 , 2 8 5 - 9 1 . Nagineni, C.N. and Bhat, S.P. (1989b). c~B-crystallin is expressed in kidney epithelial cell lines and not in fibroblasts. FEBS Letts 249, 89-94. Nishida, S. (1982). Scanning electron microscopy of the zonular fibers in human and monkey eyes. In The Structure of the Eye. (Ed. Hollyfield, ]. G.). Pp. 357-67. Elsevier North Holland, Inc. : The Netherlands. Okada, T.S., Eguchi, G. and Takeichi, M. (1971). The expression of differentiation by chicken lens epithelial in vitro cell culture. Dev. Growth Diff. 13, 323-36. Piatigorsky, J. and Zelenka, P. (1990), Transcriptional regulation of crystafiin genes: cis elements, transfactors and signal transduction systems in the lens. Adv. Dev. Biochem (in press). Reddan, J. R. and Wilson-Dziedzic, D. (1983). Insulin growth factors and epidermal growth factor trigger mitosis in lenses cultured in a serum-free medium. Invest. Ophthalmol. Vis. Sei. 24, 409-16. Reichmann, E., Ball, R., Groner, B. and Friis, R. R. (1989). New mammary epithelial and fibroblast cell clones in culture from structures competent to differentiate functionally. ]. Cell Biol. 108, 1127-38. Rothstein, H., VanWyk, J. ]., Hayden, J. H., Gordon, S.R. and Weinsieder, A. (1980). Somatomedin C: restoration in vivo of cycle traverse in GO/G1 blocked cells of hypophysectomized animals. Science 208, 410-12. Sanders, E. J. and Bouziges, F. (1988). The role of epithelialmesenchymal cell interactions in developmental processes. Biochem. Cell Biol. 66, 530-90. Takeichi, M. (1988). The cadherins: cell-cell adhesion molecules controlling animal morphogenesis. Development 102, 639-55.
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Thomson, J. A. and Augusteyn, R. C, (1985). Ontogeny of human lens crystallin. Exp. Eye Res. 40, 393-410. Wistow, G.J. and Piatigorsky, J. (1988). Lens crystallins: evolution and expression of proteins for a highly specialized tissue. Ann. Rev. Biochem. 57, 479-504. Yamamoto, Y. (1976). Growth of lens and ocular environment: role of neural retina in the growth of mouse
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lens as revealed by an implantation experiment. Dev. Growth Diff. 18, 273-8. Young, W.R. and Ocumpaugh, D.E. (1966). Autoradiographic studies on the growth and development of the lens capsule in the rat. Invest. Ophthalmol. 5, 583-93.
Lens Fiber Cell Differentiation and Expression of Crystallins in Co-cultures of Human Fetal Lens Epithelial Cells and Fibroblasts CHANDRASEKHARAM
N. N A G I N E N I *
AND S U R A J P. BHAT'~
Jules Stein Eye Institute, University of California, School of Medicine, Los Angeles, CA 90024-1771, U,S.A. (Received Bethesda 5 February 1991 and accepted in revised form 5 March 1991) Growth of the ocular lens is directed by the division and differentiation of a single layer of epithelial cells located at the equatorial region. It is conceivable that this region of the lens capsule presents a special microenvironment modulated by molecular cues emanating from the surrounding tissues. In an effort to investigate the source and nature of these molecular cues, we co-cultured human fetal lens epithelial cells and fibroblasts derived from the ciliary body. We observed morphological differentiation as evidenced by the appearance of differentiating lentoid structures associated with fibroblasts. Characterization of the expression of lens-specific proteins revealed that in addition to ~B-crystalfin, these lentoid structures contain the lens fiber cell-specific proteins, aA-crystallin, flB~-crystallin and yS-crystafiin. None of these crystallins could be found in the surrounding undifferentiated lens epithelial cells. Interestingly, aBcrystallin usually present in lens epithelial cells when cultured alone, was found to be markedly decreased, both in synthesis and content in the cells surrounding the differentiated structures, suggesting that the process of differentiation in vitro may concomitantly produce a factor(s) which modulates aBcrystallin expression in these cells. Key words: lens; epithelium; differentiation; crystallins; co-culture; fibroblasts.
1. Introduction The development of the ocular lens presents an attractive paradigm for the study of the process of differentiation and its attendant molecular components. Based on embryological investigations it has been suggested that polarity of differentiating lens fiber cells at the equatorial zone is determined by extrinsic factors (Coulombre and Coulombre, 1963). Historically, these factors have been considered to be derived from the neuroretina. This consideration has been based on the proximity of the optic vesicle to the presumptive lens ectoderm during the early phase of lens induction and the observation of differentiating posterior cells of the lens vesicle. However, in the postembryonic lens the differentiation of lens epithelial cells is spatially restricted to the equatorial region (Maisel et al., 1981) which is next to the surrounding ciliary body. It is obvious from the spatially restricted, differentiation zone that the equatorial region presents a different microenvironment than the rest of the capsule surrounding the lens. It has been assumed by a number of investigators that the special environment in this differentiating zone is modulated by molecular cues coming from the retina, diffusing through the vitreous and concentrating at the equatorial regions. This rationale has guided investigators to examine the response of lens epithelial cells to neuroretina or the * Present address: Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD 20862, U.S.A. ~" For correspondence.
00144835/92/020193 +08 $03.00/0 13
extracts~factors derived from the neuroretina. These responses have varied from proliferation of the epithelial cells to the promotion of differentiation (McAvoy, 1980; Arruti, Ciriflo and Courtois, 1985; Courty et al., 1986; Piatigorsky and Zelenka, 1990, for review). One of the first factors shown to promote differentiation in epithelial cell explants was isolated from chicken vitreous and characterized to be Somatomedin C or insulin-like growth factor (Beebe et al., 1987). Explants from the rat lens epithelium have been shown to respond to fibroblast growth factor by differentiation (Chamberlain and McAvoy, 1987). Other factors such as Somatomedin C (Rothstein et al., 1980) and PDGF (Brewitt and Clark, 1988) have been suggested to be involved in epithelial cell proliferation and lens growth, respectively. Thus, at the present time the source(s) and the type of stimuli that modulate lens differentiation remain conjectural. In this report we present investigations conducted on lens fiber cell differentiation in vitro from the perspective of cellular interactions. This approach is based on epithelial-mesenchymal interactions known to induce differentiation and specific gene expression during early development (Grobstein, 1954; Kratowil and Schwartz, 1976; Jacobson and Sater, 1988; Sanders and Bouziges, 1988; Reichmann et al., 1989). We have co-cultured h u m a n lens epithelial cells with fibroblasts. The source of fibroblasts was selected on the following basis: the equatorial region is in close juxtaposition to the ciliary body (Nishida, 1982) which has been invoked to influence lens growth within this area (Mikuficich and Young, © 1992 Academic Press Limited EER 54
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FIG. 1. Phase contrast photomicrographs of cultures of human fetal lens epithelial cells (LEC) and human fetal ciliary body fibroblasts (CBF). A, Pure population of confluent CBF. B, CBF plated at a lower density to form non-confluent cultures producing a network of connections between the ceils. C, Secondary culture of a pure population of confluent LEC. D, LEC and CBF after 4 weeks of co-culture showing the characteristic fiber network (arrows) produced by the fibroblasts. Bar = 200 #m. 1963 ; Young and Ocumpaugh, 1966). As a first step we cultured fibroblasts from the surrounding tissue (ciliary body) and then investigated the response of h u m a n fetal lens epithelial cells (Nagineni and Bhat, 1988) to co-culture with these cells. The response which manifests morphologically in differentiating fibers (lentoid bodies, Okada, Eguchi and Takeichi, 1971) was characterized by following the appearance a n d / o r disappearance of lens fiber cell-specific proteins.
2. Materials and Methods
Cell Cultures Human fetal lens epithelial cells (LEC) and pure populations of h u m a n fetal ciliary body fibroblasts (CBF) derived from h u m a n fetuses of 1 8 - 2 4 weeks of age, were cultured from the lens capsule-epithelium and ciliary body explants, respectively, as described earlier (Nagineni and Bhat, 1988, 1989a, b). Pure populations of LEC and CBF in primary culture were dissociated with trypsin-EDTA and seeded at a 9:1 (LEC:CBF) ratio for co-culture.
Analysis of Fibroblast-associated Lentoid Structures The fiber network and associated lentoid structures produced in the co-cultures of LEC and CBF were
removed easily by holding with fine forceps at one end and lifting it away. The entire fiber network was interconnected and could be peeled away leaving the undifferentiated lens epithelial cells behind. These structures were collected from five to eight 35-mm culture dishes and pooled for analysis. Supernatant fractions of human fetal lens, LEC and CBF were prepared as described earlier (Nagineni and Bhat, 1988, 1989a, b). Cell cultures or fiber network and its associated cells were extracted with lysis buffer [10 mM Tris (pH 7"4), 100 mM NaC1, 2 mM EDTA 1% NP-40, 0'5% deoxycholate and 0.5% triton X-lO0]. After freezing and thawing four times on dry ice, the lysate was centrifuged at 1 7 0 0 0 g for 20 min (Sorvall, RC5C, Dupont, Wilmington, DE) and the supernatant used for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) a n d / o r immunoblotring (Nagineni and Bhat, 1988).
Immunoblotting Proteins in the supernatant fractions were separated on 15% SDS-PAGE minigels (Hoeffer Inc., San Francisco, CA). After transferring proteins onto nitrocellulose (Schleicher and Schuell, Inc., Keene, NH), the membranes were incubated in the presence of specific rabbit-antisera to different crystallins. HRP (Horse radish peroxidase) conjugated goat anti-rabbit IgG was used as the second antibody and colour was
LENS CELL D I F F E R E N T I A T I O N
IN VITRO
developed with H202 and p-chloro 1-napthol (Biorad Laboratories, Sacramento, CA).
Labeling of Cells Fibroblast-associated lentoid structures (including the fiber network) were removed 2 days before labeling to prevent contamination of undifferentiated parts of culture dish. Labeling with pS]methionine (trans asSlabel, ICN Radiochemicals, Irvine, CA), preparation of supernatant fractions ( 1 7 0 0 0 g, 20 min) and SDSPAGE in 159/o gels were done as described above. 3. Results
Figure 1 shows the morphology of h u m a n fetal lens epithelial cell cultures and epithelial:fibroblast co-
Fro. 2: LEC co-cultured with CBF. A and B, After 10-12 weeks of co-culture showing translucent lentoid structures (*). C, Lentoid bodies (arrows) at the termini of the fiber network produced by fibroblasts. Bar = 100 #m.
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cultures. Co-cultures of LEC and CBF resulted in the appearance of a large number of lentoid bodies. Lentoid bodies (Okada et al., 1971) are the morphological manifestation of differentiation of lens epithelial cells into lens fiber cells. These in vitro differentiated structures have previously been shown to contain elongated differentiated fiber ceils and have been characterized by immunofluorescence and at the electron microscopic level (Okada et al., 1971; Hamada and Okada, 1978; Arita et al., 1990). Interestingly, in our system these translucent, differentiating structures were seen predominantly associated with fibroblasts which formed a fiber network surrounded by undifferentiated epithelial cells [Fig. I(D)]. Such structures are rarely observed when lens epithelial cells are cultured alone (Nagineni and Bhat, 1988), as shown in Fig. I(C). The fibroblasts used in this study, grown at high density, are shown in Fig. I(A), However, when these cells were grown at a lower density, they grew as a network of isolated cell masses in contact with each other [Fig. I(B)]. The results of co-culture of lens epithelial cells such as the ones shown in Fig. I(C) and fibroblasts [Figs I(A) and (B)] in a 9 : 1 ratio are presented in Fig. 1 (D) and Figs 2(A), (B) and (C). The lopsided ratio in favour of lens epithelial ceils had to be maintained in order to prevent inherently faster growing fibroblasts from taking over the entire culture dish. This ratio also helped in defining the boundaries of fibroblast growth within a monolayer of lens epithelial cells. Comparison of Fig. 1 (B) with Fig. I(D) suggests that the network in co-cultures (indicated by arrows) appears to be contributed by fibroblasts independent of epithelial cells. Figure I(D) is a 2-3-week-old co-culture in which transparent lentoid structures have just started to appear. Figures 2(A), (B) and (C) represent 10-12week-old co-cultures. Lentoid structures in different areas are seen associated with the fiber network formed by fibroblasts. The translucent, differentiated structures, which we presume are the differentiating fiber ceils derived from the lens epithelial cells, are found associated with this network [Figs 2(A), (B) and (C)]. Interestingly, these translucent lens fiber cells are also seen at the termini of these fibrous structures [Fig. 2(C)]. All of these structures show positive reactivity with 7-crystalfin antiserum as detected by immunofluorescence (data not shown). A thorough biochemical analysis of the differentiated structures associated with the fibroblasts was undertaken using antibodies specific to four different crystallins (aA, aB, fiB2, yS), representing three major immunologically distinct classes of proteins (a, fl and 7-crystallins) (Wistow and Piatigorsky, 1988 ; Bloemendal, Piatigorsky and Spector, 1989). These proteins are known to be present in terminally differentiated fiber cells (Bloemendal, 1981 ; Wistow and Piatigorsky, 1988). Alpha A-crystallin (aA) and aB-crystallin (aB) are assumed to be two subunits of the main structural protein a13-2
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le
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FIG. 3. Demonstration of lens fiber cell-specifc expression in lentoid structures associated with fibroblast generated network. The fiber network produced by CBF and its associated lentoid structures was peeled away with forceps and analysed by immunoblotting. A, Immunoblot with anti-a-crystallin (all the lanes are from the same blot, processed identically). Each lane designation is followed by the amount of protein loaded, in parentheses, le, Lens homogenate (5 #g). Lane 1, Fiber network collected from secondary cultures of LEC [5 weeks PC (primary culture), 24 weeks SC (secondary culture) co-cultured with CBF (45 #g)]. Lane 2, Fiber network collected from secondary cultures of LEC (12 weeks PC, 33 weeks SC), co-cultured with CBF (45 #g). Lane 3, CBF (80 #g). B, Immunoblot analysis with anti-a and anti-fiB 2 (this blot was first used with anti-a and then re-used with anti-fiBs; all the lanes are from the same blot). Lanes 1, 2 and 3 as in (A). Lane 4, Tertiary cultures (TC) of LEC that had about 80% of refractile elongated cells (Nagineni and Bhat, 1989a) (2 weeks PC, 3 weeks SC, 1 week TC: 12 #g). C, Immunoblot analysis with anti- 7. Lane 1, Secondary cultures of LEC (8 weeks PC, 25 weeks SC: 10/zg). Lane 2, Tertiary cultures of LEC (8 weeks PC, 25 weeks SC, 25 weeks TC: 10 #g). Lane 3, Pellet fraction of the cells in lane 2 (16 #g). Lane 4, Fiber network collected from secondary cultures of LEC co-cultured with CBF [same as in lane l in (A) and (B)] (20 #g). Lane 5, fiber network collected from secondary cultures of LEC co-cultured with CBF [same as lane 2 in (A) and (B) : 20 #g). Lane 6, CBF (80 #g). [In (C) the first lane contains molecular mass standards, same as shown in Fig. 4. Standards are not shown in (A) and (B).]
crystallin present in the lens fiber cells (see Discussion). BetaB2-crystallin (fiB2) is synthesized upon differentiation of lens epithelial cells into fibers, and 7-crystallins (7) are known to be synthesized during early growth of the lens, specifically in the fiber cells. Figure 3 shows that at least one representative of each of these classes of crystallins can be detected within the fibroblastassociated network which contains the translucent lentoid structures [Fig. 3(A) lanes 1 and 2 (aA and ~B), Fig. 3(B) lanes 1 and 2 (fiB2), and Fig. 3(C) lanes 4 and 5 (7S)]. The data presented in Fig. 3 was obtained from two different sets of cultures (lanes 1 and 2). This may explain the variations in the content of each protein detected on the immunoblots [e.g. lanes 1 and 2, in Figs 3(A) and (B) and lanes 4 and 5 in Fig. 3(C), represent two different sets of cultures]. A blot, similar to that presented in Fig. 3(A) was first used for aA immunoblotting and then re-used for analysis with
anti-fiB 2 [Fig. 3(B) lanes ] and 2]. It must be stressed that all these proteins have been well characterized and their positions in an SDS-PAGE well recognized (Bloemendal, 1981). Also, the antisera used in the experiments shown in Fig. 3 have been well characterized earlier (Nagineni and Bhat, 1988, 1989a, b; McFall-Ngai et al., 1985, 1986). The anti-~-crystallin antiserum reacts with both ~A and aB. Anti-flB~ specifically reacts with flB~ and anti- 7 reacts with different members of 7-crystallin family of proteins, all of which fall in the molecular mass range of 2 0 - 2 4 kDa. Among these proteins yS moves as a component of about 24 kDa molecular mass. Fibroblast extracts do not show reactivity with any of the antisera used here [Figs 3(A) and (B) lane 3 and Fig. 3(C) lane 6]. The data in Fig. 3 indicate that all classes of crystallins (a, fiB~ and 7S) could be detected only in the differentiating structures. It was of interest to
LENS CELL DIFFERENTIATION
IN V I T R O
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45-
compares them to the cells which have never been cocultured with fibroblasts (compare lanes 1, 2 and 7 with lanes 3, 5 and 6). Inhibition of the synthesis of ~B in undifferentiated epithelial cells stands out clearly. Furthermore, by immunoblotting with an ~B-specific antibody [Figs 4(A) and (B) lanes 3, 5 and 6], an appreciable decrease is seen in the content of this protein.
30-
4. Discussion
(A)
I
2
~
4
5
6
7
kDa
9467-
20-
~-aB
14-
(B kDa 7550392717-
FIG. 4. Inhibition of the expression of aB in undifferentiated LEC surrounding the differentiated structures. A, Autoradiogram of ~SS-proteins of epithelial cells labeled after removal of the fibroblast associated lentoid structures. Lane 1, Primary Cultures of pure population of LEC (total age in culture, 13 weeks). Lane 2, Secondary cultures (5 weeks PC, 13 weeks SC) of pure population of LEC. Lane 3, Secondary cultures of LEC (5 weeks PC, 13 weeks SC), co-cultured with CBF. Lane 4, Tertiary cultures of pure population of LEC (5 weeks PC, 4 weeks SC, 10 weeks TC). Lane 5, Secondary cultures of LEC (5 weeks PC, 13 weeks SC) co-cultured with CBF. Lane 6, Secondary cultures (5 weeks PC, 17 weeks SC) of LEC co-cultured with CBF. Lane 7, Tertiary cultures of pure population of LEC (5 weeks PC, 4 weeks SC, 15 weeks TC). Numbers on the left side of panel indicate the positions of standards. B, Immunob!ot analysis with anti-aB antibody: le, human fetal lens extract (2 #g) run on the two outside lanes of the immunoblot. The rest of the lanes correspond to lanes 1-7 in (A). Numbers on the left side of the immunoblot show positions of prestained protein markers.
ascertain the synthesis of proteins in the undifferentiated epithelial cells surrounding the fibroblast associated network. An immunoblot with anti-aA, anti-fiB 2 or anti- 7 of the proteins extracted from the undifferentiated cells did not show any reactivity (data not shown). Figure 4(A) shows an SDS-PAGE autoradiograph of proteins synthesized in these cells and
Growth and differentiation of the ocular lens has been primarily studied in the context of the effect of molecular influences supposedly originating from the neuroretina or the back of the eye. These studies have been based on the rationale that lens morphogenesis is induced by the emerging optic vesicle and that this molecular influence/interaction continues into the post-embryonic growth of the lens (McAvoy, 1980). Although the role of optic vesicle/retina in lens induction is questionable (Karkinen-Jaaskelainen, 1978: see Jacobson and Sater, 1988), it is possible that the retina exerts some influence on the adult lens (Yamamoto, 1976). A developmental and molecular basis for differentiation of lens epithelial cells into f b e r cells during early embryogenesis and in the postembryonic lens remains poorly understood. The spatial restriction of the differentiating lens fiber cells lends itself to speculation that the microenvironment in the differentiating zone m a y be modulated by molecular cues coming laterally from a tissue in the immediate vicinity of the equatorial zone. It must, however, be recognized that these two possible trophic influences, namely the neuroretina and the ciliary body, need not be mutually exclusive. It should also be noted that differentiation in cultured epithelial cells has been shown to be dependent on the culture dish substrate (Arita et al., 1990) and it has been demonstrated that the lens capsule (basement m e m b r a n e components) has a role in promoting differentiation of lentoid bodies in vitro (Muggleton-Harris and Higbee, 1987). Experiments presented here represent our initial attempts at understanding the special microenvironment offered by the equatorial region in the lens. The data presented above demonstrate that co-culture of lens epithelial cells and fibroblasts results in the appearance of lentoid bodies at the interface of the epithelial-flbroblast contact. Whether there is indeed communication between the lens and the surrounding tissues, particularly in the area of the equatorial zone and whether it is only the ciliary body fibroblasts that promote differentiation remains to be investigated. A further unknown is the embryological origin of the fibroblasts. The results, however, present a basis for systematic exploration of the effect of tissues surrounding the differentiating zone of the adult lens. The fact that translucent structures containing lensspecific proteins are generated upon co-culture of the
198
fetal lens epithelial with fibroblasts suggests that the fibroblasts are either providing a modified extracellular matrix, or a t r o p h i c factor (Grobstein, 1954; Beebe et al., 1987; Muggleton-Harris and Higbee, 1987; Jacobson and Sater, 1988; Sanders and Bouziges, 1_988; Reichmann et al., 1989) or that they physically interact with epithelial cells through surface molecules (for example Anklesaria et al., 1990; Fehon et al., 1990). We assume that the lentoid bodies are derived from the fetal lens epithelial i n this co-culture. Independent subculturing of fibroblasts over long periods of time did not show any appearance of lentoids. Alternatively, it is possible that the lentoid bodies are derived via transdifferentiation of fibroblasts and that this event is triggered by co-culture with epithelial cells. Transdifferentiation, however, is a spontaneous phenomenon associated with pleuripotent cells. Although unlikely, from an embryological perspective, the data presented above do not allow negation of this possibility. Further experiments with molecularly tagged cells will be needed to establish the source of lentoid bodies with certainty. Interesting aspects of the expression of crystalfns deserve mention. We have previously shown that the appearance of the fiber cell-specific protein flB~ was dependent on subculturing and not associated with any morphological manifestation such as the appearance of lentoid bodies (Nagineni and Bhat, 1988, 1989a). The expression of fiB 2 [Fig. 3(B) lanes 1 and 2] indicates that this protein may be activated by at least two alternative pathways, one of which is associated with morphological differentiation. It is important to point out that 7S increases postnatally in fiber cells in h u m a n lens (Thomson and Augusteyn, 1985). Other 7-crystallins are predominantly synthesized during fetal life and may represent predominant contributions of the primary fiber cells which are derived from the posterior cells of the lens vesicle during lens morphogenesis. Gamma S therefore represents a 7-crystallin synthesized in secondary fiber cells which are derived from the anterior epithelial cells. There are no posterior epithelial cells available in the lens vesicle after the sixth week of gestation (Moore, 1982), all of them having been transformed into primary fibers which are restricted to the fetal nucleus of the lens. Therefore, the observation [Fig. 3(C), lanes 4 and 5] that 7S, of all the 7-crystallins, is expressed upon differentiation in vitro by lens epithelial cells may represent a true in vivo expression potential of these cells with respect to the synthesis of the 7-crystallin group of proteins. The differentiated structures produced in the cocultures along with the fiber network can be removed leaving behind undifferentiated epithelial cells. This may be due to the differential expression of cell adhesion molecules (Takeichi, 1988). Analysis of the proteins synthesized by the undifferentiated cells revealed an almost specific inhibition of ~B synthesis in these cells. This observation is novel in that it is only
C.N. N A G I N E N I A N D S.P. B H A T
associated with the undifferentiated cells and not the differentiated ones. This is intriguing considering that lens epithelial cells, when cultured alone, always synthesize c~B [Nagineni and Bhat, 1988, 1989a, b; Fig. 3(B) lane 4, and Fig. 4 lanes 1, 2, 4 and 7]. This observation must be examined in the light of recent findings demonstrating aB to be an extra-lenticular protein present in a number of other tissues (Bhat and Nagineni, 1989; Dublin et al., 1989; Iwaki et al., 1989) suggesting that it may have other noncrystallin functions, Based on its presence in epithelial cells (Nagineni and Bhat, 1989b) and its appearance on a temporal basis in different tissues (Bhat and Nagineni, 1989) and the recent demonstration of the accumulation of this protein in 3T3 cells expressing v-mos and Ha-ras oncogenes (Klemenz et al., 1991) it may be hypothesized that this protein could have a role in differentiation of a variety of tissues. From the data presented above it is not possible to causally link differentiation and the inhibition of ~B in the undifferentiated cells. However, the fact that ~B continues to be synthesized in lens epithelial cells cultured alone indicates that either fibroblasts or the process of differentiation per se produces factor(s) which inhibit the synthesis of this protein in the cells surrounding the differentiating lens fbers. The data suggest a diffusible modulator of ~B synthesis. It is tempting to speculate that the inhibition of c~B in the adjacent cells may represent a molecular link in a scheme of events that brings about the inhibition of differentiation in the neighboring cells, a mechanism akin to that proposed for neuro-genesis in Drosophila, wherein a differentiating neuroblast within a sheet of neurogenic cells inhibits the adjacent cells from differentiating (Doe and Goodman, 1985). We believe that the differentiation at the interface of the epithelial-fibroblast cells reported here represents a paradigm for the study of the microenvironment in the equatorial region of the postembryonic lens. Clearly, further investigations are warranted to delineate the molecular nature of these interactions and whether eB has any direct role in any of these processes or is merely an associated indicator.
Acknowledgements We thank Dr Joseph Horowitz for providing c~- and fiB2crystallin antisera, Dr Bill O'Day for the human fetal lenses and Dr Dean Bok for critical reading of the manuscript. This work was supported by National Eye Institute, National Institutes of Health, Bethesda, MD (grant no. EY06044, S.P.B.). S.P.B. is a Research to Prevent Blindness William and Mary Greve International Scholar.
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