Exp. Eyr Res. (1990)

50, 475-482

Neural Cell Adhesion Molecule Retinas: Tissue Localization Role in Retina-Pigment STEVEN

J. FLIESLER”*,

GREGORY

(NCAM) in Adult Vertebrate and Evidence Against its Epithelium Adhesion J. COLEb

AND

ALICE

J. ADLER”

aBethesda Eye Institute, E.A. Doisy Department of Biochemistry and Molecular Biology, and Program in Cell and Molecular Biology, St Louis University School of Medicine, St Louis, MO 63110, U.S.A., bDepartment of Anatomy and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, U.S.A., and cEye Research Institute of Retina Foundation, Boston, MA 02114, U.S.A. (Received 24 July 1989 and accepted in revised form 5 October 1989) The presenceof neural cell adhesion molecule (NCAM) was examined in the neural retina. interphotoreceptormatrix (IPM), and retinal pigment epithelium (RPE)of adult bovine and frog eyes. Usingpolyclonal antibodiesraisedagainstadult isoformsof NCAM, Westernblot analysesrevealedthe presenceof NCAM in the neural retina, but not in the IPM or RPEof thesespecies. As a control, Western blot analysiswasusedto demonstratethe presenceof interphotoreceptorretinoid-bindingprotein (IRBP) in the IPM preparations. NCAM immunoreactivity was detected by light microscopicimmunocytochemistry primarily in the plexiform layers and nerve fibre layer of the frog retina. Minor immunoreactivity was alsodetectedin the inner and outer nuclear layers,but there was no detectable NCAM immunoreactivity in the IPM. outer segments,or RPE.Theseresultsindicate that NCAM is not a likely participant in the processof retina-RPEadhesionin the adult eye. Key words : NCAM ; adhesionmolecules; interphotoreceptormatrix ; retina ; retinal adhesion; retinal pigment epithelium.

1. Introduction The intimate apposition of the neural retina and retinal pigment epithelium (RPE) is required for the normal development and maintenance of retinal morphology and physiology. The molecular processes that promote and sustain this relationship are not understood in detail, although certain fundamental concepts concerning these processeshave been proposed (reviewed by Zauberman, 1979; Adler and Evans, 1985a; Marmor, 1989). These include: (1) ion and fluid transport, (2) general oxidative metabolism, (3) interdigitation between the apical microvilli of the RPE and the photoreceptor outer segments, and (4) involvement of glycoconjugates (e.g. glycoproteins, proteoglycans, etc.), either on the apposing surfaces of cells that border the subretinal space or within the interphotoreceptor matrix (IPM). Recently, attention has been drawn to specializeddomains of the IPM that ensheath the photoreceptor outer segments and appear to be physical connections between the RPE apical surface and the neural retina (Nicolaissen. 198 5 ; Johnson, Hageman and Blanks, 1985 ; Sameshima, Uehara and Ohba, 198 7: Hollyfield et al., 1989). There have been few reports (see Discussion) concerning the presence or distribution of ‘cell adhesion molecules’ (CAMS) in the IPM or retina. This diverse classof macromolecules is widely distributed in * For correspondence at: Avenue, St Louis. MO 63110.

00144835/90/050475+08

Bethesda U.S.A.

Eye Institute.

%03.00/O

3655

Vista

the tissuesof all vertebrates studied to date. especially in nervous tissues, and has been implicated in the processesof cell-cell recognition, tissue morphogenesis, and maintenance of tissue integrity (reviewed by Frazier and Glaser, 1979; Edelman, 1983. 1986; Damsky, Knudsen and Buck, 1984; Takeichi, 1987). The best characterized subgroup of this molecular family consists of the neural cell adhesion molecules (NCAMs). While the distribution of NCAMs in embryonic vertebrate retinas has been evaluated (see Edelman, 198 3), there is little information concerning NCAMs in the mature vertebrate retina (see Discussion). Considering the well-documented adhesional role performed by NCAMs in other tissues, and the potential role for such a class of molecules in retinaRPE adhesion, we examined the presence and distribution of NCAMs in the adult neural retina, RPE and IPM of two phylogenetically diverse species, bovine and frog. Our data suggestthat NCAMs are not constituents of either the IPM or the RPE and are not likely candidates for involvement in retina-RPE adhesion.

2. Materials and Methods Materials

Adult leopard frogs (Ram pipiens, Northern) were obtained from Kons Scientific (Germantown, WI), and maintained in cyclic lighting (12 hr light, 12 hr dark) at room temperature (2 1“C) for several weeks prior to use. Bovine eyes were obtained fresh from a local 0 1990 AcademicPressLimited

476

S. J. FLIESLER

A. STAIN lkdl

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B. NCAM

220,

94, 66, 60, 43, 36’ 30t RCRM IMIR

PCPM=BM

c. IRBP

RCRM IMIRPCPMBCBM

D. CONTROL

FIG. 1. Coomassie Brilliant Blue-stainedSDS-PAGEgel (A) and Westernblots (B-D) of bovine tissues.PanelB, rabbit antimouseNCAM; panel C, rabbit anti-bovine IRBP; panel D, control (non-immune)rabbit serum.RC,retina cytosol; RM, retina membranes ; IM, interphotoreceptormatrix : IR, IRBP; PC, RPE cytosol; PM, RPEmembranes ; BC, brain cytosol; BM, brain membranes.The anti-NCAM immunoreactivebands(lanesRM and BM, Mr % 170-180 kDa) are distinct from the anti-IRBP immunoreactivebands(lanesTM and IR, Mr x 135-140 kDa, and neither is observedin the control immunoblot. Molecular weight markers (Pharmacia): ferritin (220 kDa); phosphorylaseb (94 kDa): bovine serum albumin (66 kDa); catalase (60 kDa); ovalbumin (43 kDa): lactate dehydrogenase(36 kDa; and carbonic anhydrase(30 kDa).

slaughterhouse and maintained on ice prior to use. NCAM was purified from frozen, adult chicken brains (Pelfreez; Rogers, AR) as described previously (Cole and Glaser, 1986). Bovine interphotoreceptor retinoidbinding protein (IRBP) was prepared from IPM as previously described (Adler and Evans, 1985b ; Adler, Stafford and Slayter, 1987). An additional sample of bovine IRBP was obtained from Dr Barbara Wiggert (NEI, Bethesda, MD). Electrophoresis and Western blot reagents and materials, and protein molecular weight standards (see Fig. 2), were purchased from Bio-Rad Laboratories (Richmond, CA). Additional protein molecular weight calibration standards (see Fig. 1) were obtained from Pharmacia (Piscataway, NJ). Immobilon-P membranes were purchased from Millipore Corp. (Bedford, MA). Vectastain ABC reagent kits were purchased from Vector Laboratories, Inc. (Burlingame, CA). Bovine serum albumin (BSA, fraction V), buffer

components, protease inhibitors, and alkaline phosphatase conjugates of antibodies (goat anti-rabbit IgG and rabbit anti-goat IgG) were purchased from Sigma Chemical Co. (St Louis, MO). Dialysis membranes (Spectra/Par 2, 12-14 kDA cutoff) were obtained from Spectrum Medical Industries, Inc. (Los Angeles, CA). Whole antiserum raised in rabbit against adult rat brain NCAM (rabbit anti-rat NCAM) was obtained from Dr Richard Akeson (University of Cincinnati, Cincinnati, OH). A rabbit IgG fraction (rabbit antimouse NCAM) raised against adult mouse brain NCAM (the same immunogen as used by Hall and Rutishauser, 1985) was obtained from Dr Urs Rutishauser (Case Western Reserve, Cleveland, OH). These polyclonal antibodies cross-react with all known NCAM isoforms (see Discussion) without apparent phylogenetic specificity (Williams, Goridis and Akeson. 1985 ; Sunshine et al.. 1987). Rabbit anti-bovine IRBP

NCAM

IN VERTEBRATE

477

RETINAS

A. STAIN

Mr IkDl 200, ‘g 66,

MW

R RP IM IR N

B. NCAM

C. IRBP

200, 116, 97* 66,

31’ R RP IM IR N

R RP IM IR N

FIG. 2. Coomassie Brilliant Blue-stained SDS-PAGE gel (A) and Western blots (B, C) of frog retina (R), frog RPE (RP), frog IPM (IM), bovine IRBP (IR). and chicken brain NCAM (N). Panel B, rabbit anti-rat NCAM; panel C. goat anti-bovine IRBP. Molecular weight markers (Bio-Rad) : myosin (200 kDa) ; ,&galactosidase (116 kDa) ; phosphorylase b (9 7 kDa) : bovine serum albumin (66 kDa); ovalbumin (43 kDa): and carbonic anhydrase (31 kDa). Arrows denote positions of chicken brain NCAM isoforms (Mr z 145 and P 125 kDa).

(whole IgG fraction) serum

and the non-immune

used in the Western

blot analyses

rabbit of bovine

tissues (see Fig. 1) were prepared by Organon TeknikaCappel (Malvern, PA). Goat anti-bovine IRBP (whole IgG fraction) was a gift from Dr Barbara Wiggert. These polyclonal antibodies are immunoreactive to IRBP in a diversity of biological species (Wiggert et al., 1986; Adler et al., 1988). Non-immune rabbit serum used as a control in the immunocytochemical and Western blot analyses, normal goat serum, and biotinylated goat anti-rabbit IgG, were purchased from Vector. Preparation of tissues, tissue fractions and IPM

Retinas with attached RPE-choroids from 12 darkadapted frog eyes were incubated for 1 hr at 4°C in

20 ml of phosphate-buffered protease inhibitors

saline (PBS) containing

and chelating

agents (2 pg ml-’

leupeptin, 10 fig ml-’ pepstatin, 1 mM phenylmethylsulfonyl fluoride (PMSF), O-1 mM Na,EDTA, O-1 mM EGTA). RPE-choroids and retinas were transferred separately to microfuge tubes, quick-frozen in liquid nitrogen, and stored at -80°C prior to analysis. The PBS wash solution (containing the IPM) was centrifuged (1 hr at 100000 g, 4”C), and the supernatant fraction was dialyzed for 24 hr against three changes

(2000 ml each) of distilled water. The dialysate was lyophilized, redissolved in 1.0 ml of distilled water, and stored at -80°C prior to use. Bovine tissues, tissue fractions, and IPM were prepared essentially as described previously (Adler and Severin, 198 1; Adler and Evans, 198 Sb), using chilled PBS containing 1 mM PMSF and 0.2 TIU ml-’

S. J. FLIESLER

478

aprotinin. Bovine brain samples (containing approximately equal amounts of grey and white matter) were dissected from the cerebrum. Tissues (retina, RPE, and brain) were homogenized in chilled buffer (motordriven, Teflon-on-glass homogenizer) and then centrifuged for 15 min at 20000 g. The supernatants (crude cytosol fractions) were stored at - 80°C. The pellets were washed twice by centrifugation in PBS, and the resulting crude membrane fractions were homogenized with 1% (w/v) SDS and stored at -80°C. Electrophoresis and Western blot analysis Polyacrylamide slab gel electrophoresis (SDS-PAGE) was performed with a Bio-Rad Mini-Protean II gel system, using 7% acrylamide mini gels containing 0.1% SDS (Laemmli, 1970). Bovine samples (except IPM) contained 30-50 ,ug of protein per well: IPM contained 100 ,ug of protein (see Fig. 1). Protein loading levels for analysis of frog samples were as follows : neural retina and RPE-choroid, 180 yg each : IPM, 60,~g; IRPB, 25 ,ug; NCAM, 6 pg (see Fig. 2). Following electrophoresis, one portion of each gel was stained with Coomassie Brilliant Blue R-250 ; the companion portion was subjected to electrophoretic transfer (Western blotting)--either to nitrocellulose (Fig. 1) or Immobilon-P membranes (Fig. 2), essentially as described by Rodriguez and Fliesler ( 19 8 8 ) . Immunochemical staining of the nitrocellulose membrane blots was performed at room temperature, using TBST buffer (0.1 M Tris-Cl, pH 7.5, 0.5 M NaCl, 0.1% Tween-20) for all dilutions and rinses. Blots were incubated for 30 min with primary antibodies (rabbit anti-mouse NCAM, 1: 2 50 dilution ; rabbit anti-bovine IRBP, 1: 50000 dilution: or normal rabbit serum, 1: 2000 dilution), followed by a 30 min incubation with biotinylated secondary antibody (goat anti-rabbit IgG, at 1:20’000 dilution). Visualization was performed with Vectastain ABC reagents (avidin and biotinylated horseradish peroxidase. 1 : 10 dilutions, for 30 min), using a 15-min exposure to substrate solution (0.8 mg ml-’ diaminobenzidine (DAB), 0.015 % H,O,, and 0.4 mg ml-’ NiCl,, in 0.1 M Tris-Cl buffer, pH 7.5). Immobilon-P membrane blots were incubated overnight at room temperature in TBST containing 0.05% sodium azide and primary antibodies (rabbit anti-rat NCAM or goat anti-bovine IRBP, 1: 1000 dilution, or normal rabbit or goat serum, 1:2000 dilution), followed by a I-hr incubation with alkaline phosphatase-conjugated secondary antibodies (goat anti-rabbit IgG or rabbit antigoat IgG, 1:3000 dilution). Visualization was performed by exposure to a substrate cocktail, as described previously (Rodriguez and Fliesler, 198 8 ). Light Microscopic Immunocytochemistry Hemisected frog eyecups were immersed in Bouin’s fixative (Gray, 1954) at room temperature for 3 hr,

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rinsed with several changes of 80% ethanol, and processed for conventional paraffin embedment. Microtome sections ( 5-6 /lrn thickness) were transferred to gelatin-coated glass slides and processed for light microscopic immunocytochemistry, essentially as described by Nadji and Morales (1986). Briefly, endogenous peroxidase activity was blocked by treatment for 30 min with 050/o H,O, in 8OY) methanol. The deparaffinized, rehydrated sections were ‘ blocked ’ for 20 min with 1% BSA in Tris-buffed saline (TBS: 0.02 M Tris-Cl, pH 7.4, 0.9% NaCl). then exposed to rabbit anti-rat NCAM (1: 50 dilution, in TBS) or to non-immune rabbit serum (1 : 1000 dilution, in TBS) for 1 hr. Following exposure for 30 min to secondary antibodies (biotinylated goat anti-rabbit lgG, 1 : 1000 dilution, in TBS), sections were incubated with Vectastain ABC reagents for 45 min and developed with DAB-H,O, solution (80 mg DAB in 200 ml 05’%, H,O,) for 3-5 min at room temperature. Sections were counterstained with Harris’s hematoxylin and photographed with an Olympus Vanox photomicroscope. using a 25X oil immersion objective.

3. Results Western Blot Analysis of Bovine Tissues and PM Figure 1 shows the results obtained by SDS-PAGE and Western blotting for bovine retina, RPE, IPM and brain fractions. Membrane fractions from both neural retina (RM) and brain (BM) exhibited a component at z 170-l 80 kDa that was cross-reactive to rabbit gii-mouse NCAM antibodies [Fig. l(B)], consistent with the molecular weight of one of the known NCAM isoforms (NCAM-A: see Discussion). Since brain is a prominent source of NCAM (see Edelman, 1983). the brain membrane sample served as a positive control for anti-NCAM immunoreactivity. There were no similar immunoreactive components present in the IPM (IM), cytosol fractions of retina or brain (RC, BC), or RPE cytosol or membrane fractions (PC, PM). Furthermore, the IRBP (IR) sample did not exhibit cross-reactivity with anti-NCAM antibodies. Several of the tissue samples, but not the IPM, exhibited one or more apparently immunoreactive bands in the 5 5-6 5 kDa range. However, the control blot [Fig. l(D)] also exhibited these bands ; hence, these presumably are artifacts unrelated to authentic anti-NCAM immunoreactivity. The IPM and IRBP samples exhibited strong immunoreactivity, as expected, to anti-IRBP antibodies [Fig. l(C)], which stained primarily, a component of Mr % 13 S-140 kDa. This is consistent with the known molecular weight of IRBP (see Adler and Evans, 1985a. and references cited therein) and the Coomassie Brilliant Blue staining pattern shown for IRBP in Fig. l(A). The minor, apparently immunoreactive bands seen in other lanes of the blot are likely artifacts, since most of these are seen also in the blot treated with non-immune serum.

NCAM

IN VERTEBRATE

Western Blot Analysis of Frog Tissues and IPM The Western blots of frog whole neural retina, whole RPE-choroid, and IPM are shown in Fig. 2. Frog neural retina (R) exhibited a prominent component to (Mr z 180-190 kDa) which was immunoreactive rabbit anti-rat NCAM antibodies, as well as a smaller minor immuno(Mr z 1 SO-l 60 kDa), relatively reactive component [Fig. 2(B)]. The larger immunoreactive species had an apparent molecular weight similar to that of the anti-NCAM immunoreactive components found in bovine retina and brain membrane fractions [see Fig. l(B)]. The smaller species was similar to one of the other known NCAM isoforms (NCAM-B; see Discussion), but might simply represent a degradation product of the larger component. An authentic sample of chicken brain NCAM (N) exhibited two, approximately equal, diffuse bands of immunoreactivity in the molecular weight ranges 140-150 kDa and 120-l 30 kDa, respectively (consistent with NCAM-B and NCAM-C isoforms, respectively ; see Discussion). Frog RPE-choroid (RP) and IPM (IM) showed no cross-reactive components of similar apparent molecular weight. When these samples were blotted against anti-IRBP antibodies [Fig. 2(c)], only the lanes corresponding to the IPM and authentic bovine IRBP exhibited immunoreactivity, with a prominent band at Mr x 135-140 kDa. Only trace anti-IRBP immunoreactivity was observed in the retina and RPB-choroid samples (probably representing adherent IPM), and there was no crossreactivity with the NCAM sample. Control immunoblots (not shown) did not exhibit immunoreactive components corresponding to the molecular weights of NCAMs or IRBP. although several minor, apparently artifactual bands of lower molecular weight [similar to those shown in Fig. l(D)] were observed. lmmunocytochemical

479

RETINAS

Localization of NCAM

The localization of NCAM immunoreactivity in the adult frog retina is demonstrated in Fig. 3. The inner and outer plexiform layers were intensely immunoreactive to anti-NCAM antibodies (upper panel), as was the nerve fiber layer. The inner limiting membrane also appeared immunoreactive. Less intense, diffuse immunoreactivity was observed in the matrix surrounding the cells in the inner and outer nuclear layers. In addition, the intensity of immunoreactivity in the outer half of the inner nuclear layer appeared somewhat greater than in the inner half. However, there was no detectable immunoreactivity in the IPM, RPE, or outer segments. The control (treated with non-immune rabbit serum; lower panel) exhibited no specific immunostaining ; the brownish-black coloration in the IPM in both samples is due to melanin granules in the apical processes of the RPE. which surround the outer segments and extend almost to the level of the outer limiting membrane. This coloration

is clearly distinguished from the tannish-brown. DABperoxidase reaction product indicative of positive immunoreactivity. While we report here the results of only a single set of conditions for immunocytochemical analysis. it should be appreciated that we systematically varied the conditions (e.g. concentrations and exposure times of primary and secondary antibodies and substrates) to obtain maximum specific immunostaining. 4. Discussion Previous light microscopic immunocytochemical studies have demonstrated that NCAM is expressed in all cell layers of the embryonic chicken neural retina (Cole and Glaser, 1984; Daniloff et al., 1986) and also appears to be localized to those layers of the adult chicken retina (Daniloff et al., 1986). However, in those studies, the presence or absence of NCAM in the IPM or RPE was not clearly discernable from the authors’ micrographs. The data presented in this study demonstrate that the IPM of adult vertebrate retinas does not contain NCAM, nor is NCAM present on the surfaces of cells which border the subretinal space. Once NCAM is expressed in tissues of neuroectoderm origin (such as the neural retina and RPE), it tends to persist in those tissues, although sometimes in reduced amount (see Edelman, 1986). Considering the exquisite sensitivity of the immunodetection methods employed, it is improbable that our failure to detect NCAM in the IPM or on the associated cell surfaces was due to inadequate methodology. Therefore, it is unlikely that NCAM was originally expressed in the embryonic IPM (or on the cell surfaces bordering the subretinal space) and subsequently was reduced to undetectable levels at some later stage of development. Accordingly, our results tend to rule out NCAM as a significant participant in retina-RPE adhesion, either in the developing or adult vertebrate eye. NCAM is present in the neural retina, primarily in the plexiform layers and nerve fiber layer. There also is indication that NCAM is distributed extracellularly within the inner and outer nuclear layers. The biochemical results (especially Fig. 1) indicate that retina NCAM is membrane-bound. This is consistent with the fact that soluble NCAM isoforms in other tissues represent an extremely minor fraction of total NCAMs (Bock et al., 1987). Hence, the apparent extracellular distribution of NCAM in the retina actually reflects cell surface-associated NCAM within the various retinal ceil layers, consistent with its potential roles in the maintenance of cellular organization and functional cell-cell contacts (see Frazier and Glaser, 1979; Edelman, 1983, 1986). There are three major NCAM isoforms, based upon their apparent molecular weights (Mr) as determined by SDS-PAGE gel electrophoresis analysis : ‘NCAM-A ’ (Mr z 160-190 kDa), ‘NCAM-B’ (Mr z 130-150 kDa, and ‘NCAM-C’ (Mr z 90-120 kDa). This het-

480

erogeneity is largely due to alternative KNA processing of a single NCAM gene, resulting in expression of polypeptides of differing sizes (for a review. see Cunningham et al., 198 7). In addition, post-translational and developmentally-regulated modifications, such as changes in the z2,8-polysialic acid content of the oligosaccharide chains of NCAMs, also contribute to apparent molecular weight heterogeneity (see Edelman, 198 3 ; cf. Linnemann, Lyles and Bock, 198 5 ; Sunshine et al., 1987). Virtually all of the NCAM detectable in adult neural retina of both bovine and frog eyes appears to be the NCAM-A isoform. This isoform is also the major NCAM species present in adult Xenopusbrain (Mr z 180 kDa; Sunshine et al., 1987). In all three of these cases, lower M, isoforms (e.g. NCAM-B) are barely detectable (if at all) by Western blot analysis, suggestingthat they are present only in extremely minor amounts. Since the antiNCAM antibodies usedin the present study cross-react with all known NCAM isoforms, and since the authentic adult chicken brain NCAM isoforms transferred efficiently and were detectable under the conditions employed (seeFig. 2). failure to detect other NCAM isoforms is not likely due to limitations of technique. However, since the relative expression of NCAM isoforms in the adult nervous system is speciesdependent (seeEdelman, 198 3 ; Sunshine et al., 19 8 7), we cannot conclude, a priori, that NCAM-A is the sole isoform to be found in retinas from all adult species. It should be appreciated that we have examined only one member of the relatively expansive family of adhesion molecules. It is possible that one or more other members of this family may be present in (or in closeassociation with) the IPM and involved in retinaRPE adhesion. For example, while NCAM-mediated ceil-cell adhesion is calcium-independent(seeEdelman, 1983), the cadherins represent a class of cellassociated,calcium-dependentadhesion molecules (see Takeichi, 1987). In this regard, retina-RPE adhesion in vitro is dramatically reduced in medium lacking divalent cations (i.e. Ca”+ or Mg”+), and adhesion largely can be restored by returning the tissues to medium containing these cations (Yao, Endo and Marmor, 1989). Although the molecular basisof this cation-dependent adhesion is unknown, the potential involvement of cadherins or related molecules is worth further consideration. The initial report of the presence of fibronectin (a member of another class of adhesion molecules: see review by Damsky et al., 1984) on the apical RPE surface (Pino, 1986) was later discounted by the results of a subsequent study (Philp and Nachmias. 1987). However, more recent studies demonstrating localization of fibronectin and its receptor (integrin) in a feline model for retinal detachment and in cultured RPE cells from various animal species (Anderson, 1988; Anderson et al., 1988; Fisher and Anderson, 1989) have stimulated renewed interest in the potential involvement of these moleculesin retina-RPE adhesion. Further studies will

S. J

FLIESLER

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necessary to evaluate the presence and potential functions of adhesion molecules in the retina. be

Acknowledgements This study was supported,in part, by U.S.P.H.S.grants EY06045 (SJF),EY04368 (AJA). EYO7130(GJC),and by an unrestricteddepartmentalgrant from Researchto Prevent Blindness,Inc. (S.J.F.).We thank Drs Richard Akeson and IJrs Rutishauserfor providing the anti-NCAM antibodies. and Dr BarbaraWiggert for someof the IRBP and anti-IRBP antibodiesusedin this study. We alsothank LisaCruz and RommaSouthwickfor technical assistance, SherieKleekamp and Jim Stoutenbergfor photographic assistance.and Drs GregoryS.Hagemanand DonH. Andersonfor many helpful commentsand discussions during the courseof this work. References Adler. A. J. and Evans.C. D. ( 1985a). Proteinsof the bovine interphotoreceptormatrix : Retinoid binding and other functions.In The Interphotoreceptor Matrix itI Health and Disease (Eds Bridges,C. D. and Adler, A. I.). Pp. 65-88. Alan R. I&s. Inc.: New York.

Adler, A. J. and Evans. C. D. (1985b). Some functional characteristics of purified bovine interphotoreceptor retinol-binding 273-82.

protein. Invest. Ophthalmol.

Vis. Sci. 26,

Adler, A. J. and Severin,K. M. (198 1). Proteinsof the bovine interphotoreceptor matrix: tissues of origin. Exp. Eyr Res. 32, 755-69. Adler, A. J., Spencer, S. A., Heth, C. A. and Schmidt, S. Y. (1988). Comparison of proteins in the interphotoreceptor matrix of vertebrates. Ophthalmo!. Rrs. 20. 275-85. Adler, A. J., Stafford, W. F. and Slayter, H. S. (1987). Size and shape of bovine interphotoreceptor retinoid-binding

protein by electron microscopy and hydrodynamic analysis. 1. Biol. Chem. 262, 13198-203. Anderson, D. H. (1988). Is there a molecular basis for retinal adhesion? Proc. Znt. Sot. Eye Res. 5. 99. Anderson, D. H.. Guerin, C. J., Matsumoto, B.. Kaska, D. I). and Pfeffer. B.-A. (1988). The fibronectin receptor is present on the apical surface of cultured mammalian retinal pigmented epithelial (RPE) cells. Invrst. Ophthdmol. Vis. Sci. 29 (ARVO Suppl.), 417. Bock, E.. Edvardsen, K.. Gibson, A., Linnemann, D., Lyles. J. M. and Nybroe. 0. (1987). Characterization of soluble forms of NCAM. FEBS Lett. 225. 33-6. Cole, G. J. and Glaser, L. (1984). Identification of novel neural- and neural retina-specific antigens with a monoclonal antibody. Proc. N&l. Acad. SC?. L1.S.A. 81. 22604. Cole, G. J. and Glaser, L. (1986). A heparin-binding domain from NCAM is involved in neural ceil-substratum adhesion. 1. Cell Biol. 102. 403-12. Cunningham, B. A., Hemperly. J. J., Murray, B. A., Prediger, E. A., Brackenbury, R. and Edelman, G. M. ( 1987). Neural cell adhesion molecule : Structure, immunoglobulin-like domains, cell surface modulation. and alternative RNA splicing, Science 236, 799-806. Damsky. C. H.. Knudsen, K. A. and Buck, C. A. (1984). Integral membrane proteins in cell-cell and cellsubstratum adhesion. In The Biology of GZycoproteins (Ed. Ivatt. R. J.). Pp. l-64. Plenum: New York. Daniloff. J. K.. Chuong, C.-M., Levi, G. and Edelman, G. M. (1986). Differential distribution of cell adhesion molecules during histogenesis of the chick nervous system. 1. Neurosci. 6. 739-58.

NCAM

IN VERTEBRATE

RETINAS

FIG. 3. Light microscopic immunocytochemical localization (DAB-immunoperoxidase method) of NCAM in frog retina. Upper panel, rabbit anti-rat NCAM; lower panel, control (non-immune) rabbit serum. Note intense immunoreactivity of plexiform and nerve fiber layers, and absence of immunoreactivity of the IPM, apical RPE, and OS. RPE. retinal pigment epithelium ; OS, outer segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer: IPL. inner plexiform layer: CCL. ganglion cell layer: NFL, nerve fiber layer. x 970.

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Edelman, G. M. ( 1 Y8 3). Cell adhesion molecules. Science 219. 450-7. Edelman. G. M. (1986). Cell adhesion molecules in the regulation of animal form and tissue pattern. Annu. Rev. Cell Biol. 2, 81-116. Fisher, S. K. and Anderson, D. H. (1989). Cellular effects of detachment on the neural retina and the retinal pigment epithelium. In Retina (Ed. Ryan, S. J.). Vol. 3. Pp. 165-90. C. V. Mosby Co.: St Louis. Frazier, W. and Glaser. L. (1979). Surface components and cell recognition. Annu. Rev. Biochem. 48, 491-523. Gray, P. (19 54). The Microtomist’s Formulary and Guide. McGraw-Hill: New York. Hall, A. K. and Rutishauser. U. (1985). Phylogeny of a neural cell adhesion molecule. Dev. Biol. 110, 3946. Hollyfield, J. G.. Varner, H. H., Rayborn. M. E. and Osterfeld. A. M. (1989). Attachment of the retina to the pigment epithelium: Linkage through the conedom, an extracellular sheath associated uniquely with cone photoreceptors. In Extracellular and Intracellular Messengers in the Vertebrate Retina (Eds Pasantes-Morales. H. and Redburn. D. A.). Pp. 1-1 1, Alan R. Liss. Inc. : New York. Johnson, L. V.. Hageman. G. S. and Blanks, J. C. (1985). Restricted extracellular matrix domains ensheath cone photoreceptors in vertebrate retinae. In The Interphotoreceptor Matrix in Health and Disease (Eds Bridges, C. D. and Adler, A. J.). Pp. 3 344. Alan R. Liss. Inc. : New York. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature

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Linnemann, D., Lyles, J. M. and Bock, E. ( 19 85). A developmental study of the biosynthesis of the neural cell adhesion molecule. Dev. Neurosci. 7, 230-8. Marmor. M. F. (1989). Mechanisms of normal retinal adhesion. In Retina (Ed. Ryan, S. J.). Vol. 3. Pp. 71-87. C. V. Mosby Co.: St Louis. Nadji. M. and Morales, A. R. (1986). Immunoperoxidase Techniques: A Practical Approach to Tumor Diagnosis. American Society of Clinical Pathologists Press : Chicago.

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Nicolaissen, B.. Jr. (198 5). Connections between the sensory retina and the retina1 pigment epithelium. Actn Ophthalmol. 63, 68-72. Philp, N. J. and Nachmias, V. T. (1987). Polarized distribution of integrin and fibronectin in retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 28, 1275-Y 7. Pino, R. M. ( 1986). Immunocytochemical localization of fibronectin to the retina1 pigment epithelium of the rat. Invest. Ophthalmol. Vis. Sci. 27, 840-4. Rodriguez. I. R. and Fliesler, S. J. (1988). A 42,000-Da protein in rabbit tissues and in a glycogen synthase preparation cross-reacts with antibodies to glycogenin. Arch. Biochem. Biophys. 260, 628-37. Sameshima. M.. Uehara, F. and Ohba, N. (1987). Specialization of the interphotoreceptor matrices around cone and rod photoreceptor cells in the monkey retina, as revealed by lectin cytochemistry. Exp. Eye Res. 45. 845-63. Sunshine, J.. Balak, K.. Rutishauser. U. and Jacobson, M. (1987). Changes in neural cell adhesion molecule (NCAM) structure during vertebrate neural development. Proc. N&l. Acad. Sci. U.S.A. 84, 5986-90. Takeichi, M. (1987). Cadherins : a molecular family essential for selective cell