Fibroblast growth factor receptor deficiency in dystrophic retinal pigmented epithelium

JOURNAL OF CELLULAR PHYSIOLOGY 154:631-642 (1993)

Fibroblast Growth Factor Receptor Deficiency in Dystrophic Retinal Pigmented Epithelium F. MALECAZE, F. MASCARELLI, K. BUCRA, G. F U H R M A N N , Y. COURTOIS, AND D. HICKS* INSERM U. 118, Unite de Recherches GProntologiques, (F.M., F.Mas., K.B., C.F., Y.C., D.H.), and INSERM U . 29, Hopital Cochin (K.B.), 75016 Paris, France The retinal pigmented epithelium (RPE) is known to be the site of the primary lesion in inherited retinal dystrophy in the Royal College of Surgeons (RCS) rat, a model for retinitis pigmentosa. Although the only functional defect so far detected in these cells i s their failure to efficiently phagocytose shed photoreceptor outer segment debris, the actual cause of photoreceptor cell death is still unknown. Recently the possibility of ”trophic factors” important in photoreceptor survival produced by normal RPE but not by dystrophic RPE has been suggested. Hence we decided to investigate the presence and abundance of two candidate diffusible factors, the acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), as well as their high affinity cell surface receptors (FGF-R). mRNA was isolated from primary cultures of purified normal and dystrophic RPE and analyzed by PCR amplification using specific oligonucleotide primers for aFGF and bFGF: the size and abundance of amplified fragments was similar for both cell types. Also, aFGF protein, detected by imrnunocytochemistry using specific antisera, appeared to be present in approximately equal amounts and distributed in a similar pattern. However, scatchard analysis of radio-labelled bFGF binding to primary cultures of normal and dystrophic rat RPE revealed that dystrophic RPE possess only 29% the number of surface receptors compared to congenic normal cells. Furthermore, the level of expression of FGF-R2 mRNA, but not that of FGF-R1, was significantly different. Other parameters measured (receptor affinity, profile of ligand internalization and degradation, receptor molecular weight and mitogenic activity) did not show any significant differences between normal and dystrophic RPE. The precise role of FGF-R deficiency in the etiology of the disease hence remains to be determined. but it indicates the importance of trophic factors in the normal functioning of the retina. 1993 Wiley-Liss, Inc

The human retina is susceptible to numerous hereditable pathologies that result in the degeneration of the photosensitive photoreceptors and loss of vision. Although in certain cases mutations in specific proteins have been identified in many families (Dryja et al, 19901, we still know very little about the molecular mechanisms underlying photoreceptor cell death. Animal models of human visual disorders have been of great value in studying basic mechanisms of cellular malfunction. For example, photoreceptor degeneration in the rd mouse seems due to malfunctions in the p subunit of cGMP-phosphodiesterase, which lead to accumulation of cGMP in the p sub-unit of cGMP-phosphodiesterase (Bowes et al., 1990). Photoreceptor degeneration observed in rds mouse, in contrast, is due to defects in a structural protein, peripherin (Travis et al., 1991).Whereas in these cases the primary lesion seems to reside within the neural retina itself, the Royal College of Surgeons (RCS) rat has long been known as an example where the lesion occurs exterior to the clinically affected tissue. Studies using chimaeric rat embryos composed of normal and dystrophic cells showed clearly that the primary lesion resides within the adjacent retinal pigmented epithelial (RPE) cells, although 0 1993 WILEY-LISS, INC.

these cells themselves do not disappear (Mullen and La Vail, 1976). The RPE cells have several functions within the mammalian retina (reviewed in Bok, 1985). Forming a monolayer overlying the outer segments (0s)of the photoreceptor, they are responsible for the phagocytosis of the continually growing 0s (Young and Bok, 1969). In the latter case, the distal portions of elongating 0s are engulfed and digested by the RPE, hence removing cellular debris from the interphotoreceptor matrix (IPM). It is precisely this latter function that seems to be compromised in RCS rats, as their RPE ingest shed

Received April 23,1992; accepted October 16,1992. “To whom reprint requestskorrespondence should be addressed at Laboratoire Laveran, Clinique Ophtalmologique, Hopital Civil, 1 place de l’hopital, 67000 Strasbourg, France. F. Malecaze is now at Laboratoire d‘Ophtalmologie, CHU Rangueil, 1avenue Jean Poulhes, 31054 Toulouse, France. Parts of this work have been presented previously in abstract form at the International Retinitis Pigmentosa Association meeting, Dublin, Ireland, July 19-22, 1990.

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0s at a very reduced rate (Bok and Hall, 1971). Although this finding directly implicated the reduced phagocytic effort in the disease process, recent transplanting experiments suggest that RCS RPE may also lack some secreted trophic factor that is present in normal RPE and is important for photoreceptor cell survival (Li and Turner, 1988). The fibroblast growth factors (FGFs), originally identified a s mitogens for a spectrum of anchorage-dependent cell types and the prototype members of which are acidic and basic FGF (aFGF and bFGF, respectively) (reviewed in Gospodarowicz et al., 19871, is becoming increasingly implicated in neuronal growth and survival within the central nervous system (CNS) (reviewed in Wagner, 1991). This is also true in the retina, which constitutes a peripherally located portion of the CNS. In recent years FGF has been purified from or localized in retina, the 0s and IPM (Arruti and Courtois, 1978; Plouet et al., 1988; Hageman et al., 19911, shown to bind to basement membranes (Jeanny et al., 1987) and cell surfaces (Fayein et al., 19901, stimulate retinal regeneration from chick RPE in vivo (Park and Hollenberg, 1989), and increase photoreceptor opsin levels (Hicks and Courtois, 1988; 1992) in vitro. In recent years several high affinity FGF receptors (FGF-R) have been cloned, and a t least four families are known to exist (Partanen et al., 1992). As normal RPE are known both to synthesize FGF (Schweigerer et al., 1987) and possess FGF-R (Sternfeld et al., 1989), we decided to investigate several aspects of FGF and FGF-R activity in normal and mutant RPE cells. We demonstrate here that whereas no differences in the expression of aFGF or bFGF are detectable, dystrophic RPE have 0.05) in vitro) resemble one another, with closely correspond(Fig. 10). These observations were confirmed when the ing numbers and maximal lengths of microvilli. We data were expressed as a ratio of FGF-RZIFGF-R1 chose to use primary cultures as although this led to mRNA (Fig. 10). limitations in tissue availability, primary cultures retain many of the differentiated features of the in vivo DISCUSSION monolayer, e.g., pigmentation, polarization of apical We demonstrate here that RPE cells directly impli- and basal membranes, phagocytosis of 0s (Mayerson et cated in one form of retinal pathology while resembling al., 1984). Transcripts for both FGF-R1 and FGF-RZ are clearly their normal counterparts with respect to their expression of and response to both acidic and basic FGF do visible in the normal a s well as the diseased RPE cells, display significant differences in the number and ex- with molecular weights corresponding to the previously described major three immunoglobulin-like transcripts pression of the receptors for these growth factors. The RCS rat has long been studied a s a n animal (Partanen et al., 1992). But whereas FGF-R1 expresmodel for the group of human visual disorders collec- sion is similar between the two cell types, FGF-RZ levtively termed retinitis pigmentosa, characterized by els are significantly reduced. This decrease in FGF-R2 the gradual loss of peripheral vision, usually with early mRNA expression is not large enough to fully account

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Fig. 8. Immunolabelling of cultured normal (a)and dystrophic (d)RPE with affinity purified anti-aFGF IgG. In both cases, labelling appeared mainly in the nucleus (b and e). The signal was completely abolished in the respective cells (cand D by preincubating the antibody with 10 kg purified aFGF. Scale bar

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for the reduction in total FGF-R observed from the Braunagel et al., 1988). However, the overall composibinding studies and indicates that either expression is tion of plasma membranes from normal and dystrophic compromised in other additional classes of FGF-R rats is similar, arguing against a generalized decompoandlor that there may exist recycling or delivery prob- sition. In addition, the cells are removed from young lems whereby insufficient numbers of receptors are in- rats prior to the morphological changes following disserted into the membrane, or altered receptor occu- ease onset and are not adversely affected by the cellular pancy whereby a pool of FGF-R are not accessible to decay and release of acid phosphatases occurring later. Owing to this large difference in FGF-R number beligand binding. It is possible that the dramatic reduction in FGF-R in tween normal and mutant RPE, we decided to investithe dystrophic RCS rat RPE is a result of some other gate whether other aspects of the FGFIFGF-R system degenerative event or lesion or morphological modifica- were defective. The remaining population of RCStion. Apart from their phagocytic defect, RCS RPE rdy-p+ RPE FGF-R appear to function normally in that: plasma membranes have also been reported to differ (1)both strains respond approximately equally to exogfrom noi-ma1 RPE in their content of Na' K' ATPase, enous aFGF and bFGF in dose-response studies, with 5' nucleotidase, fatty acids and specific high molecular ED,,'s of 10.0 and 10.8 ngiml for aFGF and 1.0 and 1.5 weight membrane proteins (Clark and Hall, 1986; ngIml for bFGF, for normal and mutant RPE, respec-

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FGF-Rl

FGF-R2

FGF-R?/FGF-Rl

Pig. 10. Representative densitometric quantification of FGF-R1 and FGF-R2 mRNA in cultured RPE of normal (N) and dystrophic (D)rats. Quantification o f the optical densities are processed as described in Materials and Methods. Mean values 2 SD of dystrophic rat RPE are expressed as a percentage of normal cells, defined as 100%."P < 0.05.

Fig. 9. Northern blot analysis of FGF-R1 and FGF-R2 mRNA in RPE cell cultures of normal (N) and dystrophic (D) rats. mRNA was extracted and processed as described in Materials and Methods. RNA blots were either hybridized with FGF-R1 or FGF-R2 probes corresponding to the total extracellular region of both receptor types. The bottom panel shows the ethidium bromlde stained and photographed 28s rRNA. The positions of 18s and 28s rRNA markers are indicated on the right; the positions of a n RNA marker ladder (BRL) are indicated on the left (in kb). Signal hybridizations are observed at 4.2 kb and 4.4 kb for FGF-RL and FGF-R2, respectively.

tively. Although certain parameters such as final cell density were somewhat less for dystrophic cells, this was not statistically significant. This result is maybe somewhat surprising given the paucity of FGF-R in mutant RPE, but by analogy with other systems, e.g.,

hormone receptors, it is known that only a fraction of receptors need to be occupied to elicit a cellular response (Catt and Dufau, 19731, and thus even with their reduced complement these cells can respond to exogenous FGF. (2) Binding affinities are similar in both strains (-60 pM), indicating that no major modifications have occurred in the sites for glycosylation (which are important for FGF binding; Feige and Baird, 19881, or ligand binding domains. (3) Ligand-receptor cross-linking reveals two similar MW entities in both strains, indicating that no gross alterations in receptor size have occurred. The estimated FGF-R M W s correspond to published values for other cell types, e.g., BHK-21 fibroblasts (Neufeld and Gospodarowicz, 1986). (4)FGF internalization and degradation occur a t similar rates and with similar profiles in both strains. This pattern also resembles that observed for other cell types (e.g., Mascarelli e t al., 1991) and hence there is no defect in the ligand-receptor internalization pathway. This demonstrates that although phagocytosis of 0s is diminished in the RCS rat, the RPE cells are capable of normally internalizing molecules such as FGF. The idea has evolved that endocrine tissues possess excess receptors in order to concentrate effects of and increase the likelihood of stimulation by circulating hormones (Catt and Dufau, 1973). If such a theory can be extended to the RPE, then if reduced receptor numbers are a causal factor in this pathology, it can be argued that the diminished population leads to a n inability to generate a cellular response under physiological conditions. Thus even though mutant RPE in vitro can respond to exogenous FGF as equally well as normal cells, this may not reflect the actual environment in vivo, where receptor activation would be modulated by such parameters as receptor density, ligand concentration, or distribution of local low affinity binding sites. Such a model would not require differences in the levels of FGF(s) either within the RPE (if the mode of action is autocrine, as has been recently demonstrated for fibroblasts; Mignatti et al., 1991) or the neural retina (if the mode of action was paracrine. Soluble retinal factors have been shown to influence RPE metabolism; Pautler and Beezley, 1990). We cannot explain why, if FGF-R numbers are reduced, RCS RPE cells themselves seem to survive in this pathology. At present we can only speculate that the release of trophic factors by

DYSTROPHIC RETINAL PIGMENTED EPITHELIUM

the RPE vital for photoreceptor survival is regulated by activation of FGF-R on the RPE themselves, but that RPE survival is regulated in some other manner. Other pathological conditions exist in which a reduction in growth factor receptor numbers has been implicated in the disease process. For example, i t has been observed that reductions in the level of binding of lZ5Iinsulin of the order of 50-96%, to their receptors (figures that correspond well to the reduction in '"'1-bFGF binding estimated from Scatchard analyses of -70% in the present study) are correlated with severe insulin resistance in type B syndrome of insulin resistance (Grigorescu et al., 1987) and lipoatrophic diabetes (Kriauciunas et al., 1988). In the latter case insulin receptor mRNA levels paralleled the observed decreases in binding. We have only considered the high affinity FGF-R in this study. As RPE possess cell surface (Turksen et al., 1985) and basement membrane (Jeanny et al., 1987) heparan sulfate proteoglycan, which is known to bind FGF at low affinity (Vigny et al., 1988), there may also be modifications in these molecules in RCS RPE. Interestingly, it has been recently demonstrated that both high and low affinity FGF-R are required for correct cellular stimulation by exogenous FGF (Yayon et al., 1991). It has already been reported that differences in normal and mutant human RPE heparan sulfate exist (Hewitt and Newsome, 1988) and coupled with high affinity FGF-R deficiencies, FGF regulating systems might be impaired. In summary, this is the first report of a growth factor receptor deficiency specifically located in mutant RPE cells. I t will be of great interest to examine FGF and FGF-R expression in normal and pathological outer retina to determine the interactions and importance of these molecules in in vivo retinal development and physiology. Definitive demonstration of the involvement of FGF-R in this pathology will require tranfection of RCS RPE with vectors containing FGF-R mRNA; such studies are currently in progress.

ACKNOWLEDGMENTS The authors thank the following: Dr. H. Prats (INSERM U 168) for the generous gift of recombinant bFGF; Dr. N. Fasel for the FGF-R1 cDNA; Dr. R. Breathnach for the FGF-R2 cDNA; Dr. L. Oliver (INSERM U 118) for affinity purified aFGF antibody; A. Bouterie and M. Hartmann for technical assistance; H. Coet for photographic work; and M.T. Guilhem for typing the manuscript.

LITERATURE CITED Arruti, C., and Courtois, Y. (1978) Morphological changes and growth stimulation of bovine epithelial cells by a retinal extract in vitro. Exp. Cell Res., 117,283-292. Bensaid, M., Malecaze, F., Prats, H . , Bayard, F., and Tauber, J-P. (1989)Autocrine regulation of bovine retinal capillary endothelial cell (BREC). Proliferation by BREC-derived basic fibroblast growth factor. Exp. Eye Res., 48:801413. Bok, D. (1985) Retinal photoreceplor-pigment epithelium interactions. Invest. Ophthalmol. Vis. Sci., 26rl659-1694. Bok, D., and Hall, M.O. (1971) The role of the uinment euithelium in the etiology of inherited retinal dystrophy i; [he rat. i.Cell Biol., 49,664-682. Bowes, C., Li, T., Danciger, M., Baxter, L.C., Applebury, M.L. and Farber, D.B. (1990) Retinal degeneration in the rd mouse is caused

641

by a defect in the (3 sub-unit of rod cGMP phosphodiesterase. Nature, ,3473377-680. Braunagel, S.C., Organisciak, D.T., and Wang, H.M. 11988) Characterization of pigment epithelial cell plasma membranes from normal and dystrophic rats. Invest. Ophthalmol. Vis. Sci., 29r10661075. Catt, K.J., and Dufau, M.L. (1973) Spare gonadotrophin receptors in rat testis. Nature New Biol., 244r219-221. Chirgwin, J.M., Pryzyla,A.E., McDonald,R.J. andRutter, W.J. (1978) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclcase. Biochemistry, 18t5294-5295. Clark, V.M., and Hall, M.O. (1986) RPE cells surface proteins in normal and dystrophic rats. Invest. Ophthalmol. Vis. Sci., 27,136145. Connolly, S., Hjelmeland, L.; and La Vail, M.M. (1991) Localization of bFGF in developing retinas of normal and RCS rats. Invest. Ophthaltnol. Vis. Sci. fsuppl.l,32(41:754. Dryja, T.P., McGee, T.L., Reichel, E.: Hahn, L.B., Cowley, G.S., Yandell, D.W., Sandberg, M.A., and Berson, E.L. (1990) A point rnutation of the rhodopsin gene in one form of retinitis pigrnentosa. Nature, 343:36&366. Edwards, R.B. (1977) The isolation and culture of rat retinal pigmented epithelium. In Vitro, 1.?:301-304. Faktorovich, E.G., Steinberg, R.H., Yasumura, D., Matthes, M.T., and La Vail, M.M. (1990) Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor. Nature, 34 7:83-86. Fasel, N., Bernard, M., Deglon, N., Rousseau, M., Eisenberg-Bron, C., and Cohen, G.H. (1991) Isolation from mouse fibroblasts of a cDNA encoding a new form of the fibroblast growth factor receptor (flg). Biochem. Biophys. Res. Comm., 178% Fayein, N., Courtois, Y., and Jeanny, J-C. (1990) Ontogeny of basic fibroblast growth factor binding sites in mouse ocular tissues. Exp. Cell Res., 188,7538. Feige, J.J., and Baird, A. (1988) Glycosylation of the basic fibroblast growth factor receptor. The contribution of carbohydrate to receptor function. J . Biol. Chem., 263:14023-14029. Gospodarowicz, D., Ferrara, N., Schweigerer, L., and Neufeld, G. (1987) Structural characterization and biological functions of fibroblast growth factor. Endocrine Rev., 8r95-114. Grigoreseu, F., Herzberg, V., King, G., Meitas, M., Elders, J., Frazer, T., and Kahn, C.R. (1987)Characterization of binding and phosphorylation defects of erythrocyte insulin receptors in the type A syndrome of insulin resistance. d. Clin. Endocrinol. Metab., fi4r549556. Hageman, G.S., Kirchoff-Rempe, M.A., Lewis, G.P., Fisher, S.K., and Anderson, D.H. (1991) Sequestration of basic fibroblast growth factor in the primate retinal inter-photoreceptor matrix. Proc. Natl. Acad. Sci. USA, 88:67606-67610. Hargrave, P.A., and O'Brien, P.d. (1991)Speculations on the molecular basis of retinal degeneration in retinitis pigmentosa. In: Retinal Degenerations. R.E. Anderson, J.G. Hollyfield, and M.M. La Vail, eds. CRC Press, Boca Raton, FL pp, 517-528. Hewitt, A.T., and Newsome, D.R. (1988)Altered proteoglycans in cultures of human retinitis pigmentosa retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci., 29:720-726. Hicks, D., and Courtois, Y. (1988)Acidic fibroblast growth factor stimulates opsin levels in retinal photoreceptor c e h in vitro. FEBS Lett., 234:475-479. Hicks, D., and Courtois, Y. (1992) Basic fibroblast growth factor stimulates photoreceptor differentiation in vitro. J. Neurosci., 12r20222033. Hicks, D., Bugra, K., Faucheux, B., Jeanny, J.C., Laurent, M., Malecaze, F., Mascarelli, F., Raulais, D., Cohen, Y., and Courtois, Y. (1991) Fibroblast growth factors in the retina. In: Prog. Ret. Res., Vol. 11. N.N. Osborne and G.J. Chader, eds. Pergamon Press, Oxford, UK, pp. 333-374. IIoussaint, E., Blanquet, P.R., Champion-Arnaud, P., Gesnel, M.C., Torriglia, A., Courtois, Y., and Breathnach, R. (1990) Related fibroblast growth factor receptor genes exist in the human genome. Proc. Natl. Acad. Sci. USA, 87t818lL8184. Jacquemin, E., Halley, C., Alterio, J., Laurent, M., Courtois, Y. and Jeanny, J-C. (1990) Localization of acidic fibroblast growth factor (aFGF)mRNA in mouse and bovine retina by in situ hybridization. Neurosci. Lett., llfir33-28. Jeanny, J.C., Fayein, N., Moenner, M., Chevallier, B., Barritault, D., and Courtois, Y. (1987) Specific fixation of bovine brain and retinal acidic and basic fibroblast growth factors to mouse embryonic eye basement membranes. Exp. Cell Res., 171t63-75. Kriauciunas, K.M., Kahn, C.R., Muller-Wieland, D., Reddy, S.S.K.

642

MALECAZE ET AL

and Taub, R. (1988) Altered expression and function of thc insulin receptor in a family with lipoactrophic diabetes. J. Clin. Endocrinol. Metab., 67r1284-1293. Li, L., and Turner, J.E. (1988) Inherited retinal dystrophy in the RCS rat: Prevention of photoreceptor degeneration by pigment epithelial cell transplantation. Exp. Eye Res., 47r911-917. Malecaze, F., Mathis, A., Arne, J-L., Raulais, D., Courtois, Y., and Hicks, D. (1991) Localization of acidic fibroblast growth factor in proliferative vitreoretinopathy. Curr. Eye Res., IOr719-729. Mascarelli, F.,Raulais, D., and Courtois, Y. (1989)FGF phosphorylation and receptors in rod outer segments. EMBO J.,8.2265-2273. Mascarelli, F., Tassin, J.,Courtois, Y. (1991)Effects of FGF's on adult bovine Miiller cells: Proliferation, binding and internalization. Growth Factors, 4r81-95. Mayerson, P., and Hall, M.O. (1986) Rat retinal pigment epithelial cells show specificity of phagocytosis. J. Cell. Biol., 103:299-312. Mignatti, P., Morimoto, T., and Rifkin, D.B. (1991) Basic fibroblast growth factor released by single, isolated cells stimulates their migration in a n autocrine manner. Proc. Natl. Acad. Sci. USA, 88rl1007-11011. Moenner, M., Chevallier, B., Badet, J., and Barritault, D. (1986) Evidence and characterization of the receptor to eye-derived growth factor 1, the retinal form of basic fibroblast growth factor, on bovine epithelial lens cells. Proc. Natl. Acad. Sci. USA, 83r5024-5028. Mullen, R.J., and La Vail, M.M. (1976) Inherited retinal dystrophy: primary defect in pigment epithelial cells determined with experimental rat chimeras. Science. 192.7994401. Neufeld, G., and Gospodarowicz, D. (1986) Basic and acidic fibroblast growth factors interact with the same cell surface receptors. J. Riol. Chem., 2615631-5637. Park, C.A., and Hollenberg, M.J. (1989) Basic FGF induces retinal regeneration in vivo. Dev. Biol., 134.201-205. Partanen, J., Vainikka, S., Korhonen, J.,Armstrong, E., and Alitalo, K. (1992) Diverse reeeptors for fibroblast growth factors. Prog. Growth Factor Res., 4r69-83. Pautler, E.L., and Beezley, D. (1990) The effects of retina-derived

factors on the electrical and ultrastructural properties of the bovine pigment epithelium. Curr. Eye Res., Yt617-625. Plouet, J., Mascarelli, F., Loret, M.D., Faure, d-P., and Courtois, Y. (1988) Regulation of eye derived growth factor binding to membranes by light, ATP or GI'P in photoreceptor outer segments. EMBO J., 7.373-376. Schweigerer, L., Malerstein, B., Neufeld, G., and Gospodarowicz, U. (1987)Basic fibroblast growth factor is synthesized in cultured retinal pigment epithelial cells. Biochem. Biophys. Res. Comm., 143t93P940. Sternfeld, M.D., Robertson, J.E., Shipley, G.D., Tsai, J., and Robertson, J.T. (1989) Cultured human retinal pigment epithelial cells express basic fibroblast growth factor and its receptor. Curr. Eye Res., 8r1029-1037. Travis, G.H., Sutcliffe, J.G., and Bok, D. (1991) The retinal degeneration slow lrds) gene product is a photoreceptor disc membrane-associated glycoprotein. Neuron, 6%-70. Turksen, K., Aubin, J.E., Sodek, J., and Kalnins, V.I. (1985)Changes in distribution of laminin, fibronectin, type IV collagen and heparan sulfate proteoglycan during colony formation by chick retinal pigment epithelial cells in vitro. J . Histochem. Cytochem.,33r66.5-671. Vigny, M., Olivier-Hartmann, M.P., Lavigne, M., Fayein, N., Jeanny, J-C., Laurent, M., and Courtois, Y. (1987) Specific binding to fibroblast growth factor to basement membrane-like structures and to purified heparan sulfate proteoglycan of the EHS tumor. J. Cell. Physiol., 1 3 7 . 3 2 4 2 8 . Wagner, J.A. (1991)The fibroblast growth factors: an emerging family of neural growth factors. Curr. Topics Microbiol. Immunol., 165:95-118. Yayon, A,, Klagsbrun, M., Esko, J.D., Leder, P., and Ornitz, D.M. (1991) Cell surface heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell, 64r84 1-848. Young, R.W., and Bok, D. (1969) Participation of the retinal pigment epithelium in the rod outer segment renewal process. J. Cell Biol., 42r392403.