Developmental and biochemical studies of adhesive specificity among embryonic retinal cells

DEVELOPMENTAL BIOLOGY 69, 108-117 (1979)

Developmental and Biochemical Studies of Adhesive Specificity among

Embryonic Retinal Cells

ROBERT CAFFERATA,’ JEFFREY PANOSIAN, AND GLADYS BORDLEY

Department of Biology, University of Rochester, Rochester, New York 14627

Received July 31, 1978; accepted in revised form September 29, 1978

One pattern of cell-cell adhesion within the chick embryo neural retina follows a dorsoventral gradient in which cells at either end show maximal affinity for each other. Developmental and biochemical approaches have been applied to analyze the basis of this adhesive pattern. When retinal cells were prepared from eyes that had been inverted 180” in situ prior to retinal differentiation, an inverted pattern of adhesive preference resulted. These data suggest that adhesive preference is determined early in embryogenesis. Trypsinization of either dorsal or ventral retinal cells destroyed their adhesive preference. Treatment with neuraminidase resulted in a differential loss of adhesive preference by dorsal retinal cells. This effect could be mimicked by mild oxidation with NaI04. Since periodate inactivation of adhesive preference could be reversed by subsequent borohydride treatment, borotritiate was used to label those cell surface molecules crucial to the reactivation of adhesive preference. Fluorographs prepared after polyacrylamide gel electrophoresis revealed that periodate stimulated the appearance of a small number of radioactively labeled bands. These data suggest that adhesive preference is mediated by glycoconjugates, possibly sialoglycoproteins, on the dorsal cell surface.

INTRODUCTION

One of the unsolved questions in the development of the nervous system is how growing axons recognize appropriate synaptic targets. This problem has been exten- sively studied for the central retinal projec- tions of lower vertebrates, especially among the neurons connecting spatially complementary regions of retina and optic tectum (Jacobson, 1976). Sperry (1963) proposed that complementary “cytochemical affini- ties” are distributed in map-like order across retina and tectum and these interact to establish complementarity in the pattern of synaptic connections. Recently, it has been demonstrated that embryonic cells isolated from dorsal and ventral regions of the neural retina express adhesive properties consistent with such a complementarity rule (Barbera et al., 1973; Barbera, 1975; Gottlieb et al., 1976; McClay et al., 1977). One way to examine the analogy between

’ Present address: Center for Brain Research, Uni- versity of Rochester, Box 605 Medical Center, Roch- ester, New York 14642.

these results and Sperry’s hypothesis is to map the distribution of molecules related to complementary adhesive preference. Un- fortunately, these molecules remain uni- dentified. Recent evidence suggests that adhesive preference, measured between retinal cells and complementary halves of tec- turn, depends on protein-carbohydrate interactions (Marchase, 1977).

In our experiments, the neural retina was divided into dorsal and ventral halves and single cells were dissociated from each half. Instead of assaying the adhesion of these cells to topographically related regions of visual brain, we measured their relative

adhesion to each other. This measurement provides a direct index of complementary adhesive preference, since the most stable cell-cell adhesions form between dorsoventral combinations of retinal cells and not between cells from the same half-retina (Gottlieb et al., 1976). In this report we describe developmental studies suggesting that complementary adhesive preference is determined early in embryogenesis and bio-

108 0012-1606/79/030108-10$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

CAFFERATA, PANOSIAN, AND BORDLEY Embryonic Retinal Cell Adhesive Specifici@ 109

chemical studies suggesting that it is mediated by carbohydrate residues on dorsal cells.

MATERIALS AND METHODS

Dorsal and ventral halves of neural retina were removed aseptically from lo-day embryos (stage 36/37 of Hamburger and Ham- ilton, 1951) of White Leghorn chicks (SPA- FAS). In these dissections, the choroid fissure was oriented at 6 o’clock.

The assay of cell adhesion is adapted from the cell-layer collecting technique of Walther et al. (1973) essentially according to the modification of Gottlieb and Glaser (1975). Aliquots of “‘P-labeled cells are incubated over cell layers plated in vials. The number of adhering cells is measured by quantitating the radioactivity retained by a vial after washing off nonadherent cells.

Where indicated, Hepes’ was present at 20 mM and the solution was buffered to pH 7.3. All centrifugations were at 170g for 5 min at 4°C.

Preparation of ,““P-labeled cells. Tissue from four to six half-retinas was incubated for 3 hr at 37°C in Liebowitz medium L15 (GIBCO) supplemented with Hepes, 10% (v/v) FCS, penicillin, streptomycin, and 2 mCi carrier-free “‘P (Amersham-Searle). Tissues were dissociated in 4 ml ice-cold disaggregation medium (CMF, Hepes, 10% FCS, and 50 pg/ml DNase I) by pipetting through a 22-gauge needle. Cells were centrifuged and resuspended in appropriate media. These procedures yield suspensions of predominantly single cells with an aver- age of 0.1 cpm/cell.

Preparation of cell layers. Tissue from 10 eyes was incubated for 20 min at 37°C in 5.5 ml disaggregation medium, chilled, and dissociated as above. Cells were centrifuged and washed in CMF + Hepes. Cell layers

.’ Abbreviations used: Hepes, 4-(2-hydroxethyl)-l- piperazineethanesulfonic acid; FCS, fetal calf serum (heat inactivated); CMF, Ca”‘-, Mg’+-free Hanks bal- anced salts solution; BSA, bovine serum albumin; *DR and *VR. “P-labeled dorsal and ventral retinal cells, respectively.

were plated by centrifuging 3-4 x 10” cells per derivatized vial.

Derivatization of vials. Nitric acid- washed glass vials (12-mm diameter) were reacted with 1% aqueous 3-aminopropyl- triethoxysilane (Aldrich Chemical CO.) for 3.5 hr at 100°C. Subsequent reaction with glutaraldehyde, according to the method of Gottlieb and Glaser (1975), was omitted. Before use, vials were washed in distilled water and air-dried.

Measurement of cell adhesion. To start an assay, the medium covering each cell layer was replaced with a 0.35-ml aliquot of l-4 x 10” labeled cells in assay medium (CMF, Hepes, and 0.5% BSA). Vials were incubated in a reciprocal water-bath shaker at 37°C (or O’C!) and 85 rpm. To terminate an assay, nonadherent cells were aspirated and each cell layer was rinsed three times with 0.5 ml CMF (maintained at the tem- perature of the incubation). Adherent cells were solubilized in 0.3 ml 1% Triton X-100 (Calbiochem) and radioactivity was counted in 3 ml Aquasol- (New England Nuclear). The percentage of cells adhering was calculated by dividing the cpm remain- ing in each vial by the cpm in cells at the start of the assay. We established by appropriate control experiments that this measurement was not affected by the leakage of isotope during incubation and its subsequent uptake by the cell layer (see legend to Fig. 1).

Graft procedures. Right eyes were excised, inverted, and reimplanted int,o right, orbits of the same 2-day embryos (Fig. 3). These operations required cutting a win- dow in the egg and removing the vitelline membrane. Of 23 attempts, 2 animals developed to 10 days with an inverted choroid fissure (12 o’clock). In both cases, the op- erated eye was smaller than normal and lacked an optic nerve.

Chemical treatments of”‘P-labeled cells. After each treatment, cells were centrifuged and resuspended in assay medium. Trypsin: 2 X 10’ cells/ml were incubated in CMF + Hepes containing 0.025g crystalline (3~)

110 DEVELOPMENTAL BIOLOGY VOLUME 69, 1979

-VR;DR -*VR

b A I I I I I

IO io 30 40 50 IO 20 30 40 50

TIME (MINUTES)

'VR

./--. DR r / VR

- /'

/&

I I I I I I I 1 I I 20 40 60 80 100 20 40 60 80 100

TIME (MINUTES) FIG. 1. Kinetics of dorsal and ventral cell adhesion at 37°C (A) and 0°C (B). (*DR, *VR) 32P-Labeled cell

suspensions; (DR, VR) cell layers; (-DR, -VR) vials in which the indicated cell layers were removed prior to incubation with labeled cells; (-*DR, -*VR) cell-free supernatants obtained from 32P-labeled cells following a 45-min preincubation at 37°C. This control measures the transfer of soluble label from the cell suspension to the cell layer.

trypsin (Miles) for 30 min at 37°C before Preparation of borotritiate-labeled cells. the addition of trypsin inhibitor (Sigma). After suspending 2 x lo7 cells in 1 ml 0.9% Neuraminidase: 2 x lo6 cells/ml were in- NaCl + Hepes, the following sequence of cubated in 0.9% NaCl + 10 n-&f 2-(N-mor- incubations was used: (i) 0.5 mJ4 NaBHa, pholino)ethanesulfonate (pH 6.0) contain- 20 min at O’C; (ii) NaI04 at the concentra- ing C. perfringens neuraminidase (Worth- tions indicated in Fig. 6, 10 min at 0°C; (iii) ington) for 30 min at 37°C. Periodate: lo7 4 PCi NaBT4 (Amersham-Searle), 20 min cells/ml were incubated in 0.9% NaCl + at 21’C. Cells were washed by centrifuga- Hepes containing sodium metaperiodate tion between steps. After tritiation, the (Sigma) for 10 min at 0°C (dark) before the washing medium contained 0.5 miVfNaBH4. addition of 5 mM o-n-glucose. Gel electrophoresis and autoradiofluo-

CAFFERATA, PANOSIAN, AND BORDLEY Embryonic Retinal Cell Adhesive SpecificLty 111

rography. Borotritiate-labeled cells were boiled for 3 min in the sample buffer de- scribed by Laemmli (1970). Lysates were separated by electrophoresis at 35mA constant current in slab gels of 7.5% polyacrylamide (Laemmli, 1970). Tritium was de- tected in dried gels by fluorography (Bon- ner and Laskey, 1974) with presensitized (Laskey and Mills, 1975) Kodak SB-5 film. Fluorograms were traced on a Joyce-Loebl densitometer and the relative levels of two bands, GP90 and GP40, were estimated us- ing a DuPont Curve Analyser.

RESULTS

Complementary adhesive preference. Suspensions of single cells prepared from ‘“P-labeled dorsal and ventral neural retina were tested for adhesion to cell layers de- rived from these tissues. Combinations involving labeled dorsal cells and ventral cell layers (*DR and VR) or labeled ventral cells and dorsal cell layers (*VR and DR) showed the maximal rate of adhesion (Gottlieb et al., 1976; Fig. 1A). This result occurred at both 37 and 0°C (Fig. 1B). More than 60% of *DR or *VR cells follow this

adhesive pattern, since unabsorbed cells from an initial (45-min) incubation adhere with identical kinetics when transferred to new cell layers (data not shown). Cells isolated from the same half of retina also showed measurable adhesion.

That retention of radioactivity was attributable to cell-cell adhesion was demonstrated by the fact that removal of cells from either the labeled suspension or the layer reduced labeling and abrogated adhesive preference (Fig. 1A). Cell-cell adhesion was confirmed by incubation of cell layers with suspensions of cells containing horseradish peroxidase and microscopic ex- amination of the distribution of benzidine blue (Mesulam, 1976)-stained cells (Fig. 2).

Embryonic stability. In a series of 2-day embryos the right optic primordium was excised, rotated 180”, and reimplanted so it would differentiate an inverted retina (Fig. 3). Inverted grafts, which did not derotate (Goldberg, 1976), developed a dorsal choroid fissure. These experiments were not attempted prior to stage 12 because only ventral fissures develop and these could not be distinguished from grafts which had der-

FIG. 2. Adhesion of horseradish peroxidase (HRP)-containing cells to a cell layer. Ventral cells, prepared from tissues incubated in medium L15 containing HRP, were incubated with dorsal cell layers for 20 min at 37°C. After fixation in 1% glutaraldehyde, the benzidene procedure (Mesulam, 1976) was used to stain HRP- containing cells an intense blue. Stained cells, either singly or in small clusters, always overlie the unstained cells in the layer. The fidelity of intercellular adhesion was comparable for incubations involving the other three combinations of labeled cells and cell layers. Cell-free patches appear after washing off loosely adhering cells. Phase-contrast photomicrograph was taken near the center of the vial. Bar = 50 pm.


P +-

A

180’

V D FIG. 3. Diagram illustrating the inversion of optic primordia. Only the dorsoventral axis was rotated 180’;

the anterioposterior axis remains constant.

otated. Table 1 summarizes the adhesive response of lo-day labeled cells. Inversion at stage 14 resulted in an inverted pattern of adhesive preference. This result was also obtained with labeled cells from an inversion at stage 12/13 (data not shown). These data suggest that a stable dorsoventral axis is established within the optic primordium by stages 12-14 and this axis orients the adhesive polarity of differentiating retinal cells. It was not feasible to prepare suffi- cient labeled cells to decide if optic primordia express complementary adhesive preference.

TABLE 1

EFFECT OF INVERTING THE DORSOVENTRAL OPTIC AXIS AT STAGE 14 ON CELL ADHESION”

Labeled cells

% Cells adhering to cell layers?

Normal Inverted

*DR DR 19.6 + 1.5 (6)’ 29.8 + 1.4 (6) VR 28.7 f 3.1 (6) 19.9 -t 4.7 (4)

*VR DR 27.8 f 2.0 (6) 17.6 f 1.1 (5) VR 19.9 * 1.3 (4) 26.4 f 0.8 (4)

Chemical alteration. Chemical procedures which modify cell surface components were examined for effects on adhesion. In each experiment, half of the labeled cells were treated with reagent and the other half left untreated. Adhesion was tested in the absence of reagent. By the use of a trypan blue exclusion test and by mon- itoring the release of 32P, we could show that the cells recovered from each treatment remained intact.

“Labeled cells were prepared from normal (left) and inverted (right) retinas removed from the same lo-day embryo. In the dissection of the inverted eye, the choroid fissure was oriented at 12 o’clock.

b Incubation was for 45 min at 37°C. ‘SE of the number of determinations shown in

parentheses.

TABLE 2

EFFECT OF TRYPSIN TREATMENT ON CELL ADHESION

Labeled cells

% Cells adhering to cell layers”

Control Trynsin

*DR DR 11.3 + 0.7” 9.8 f 0.1 VR 17.9 rfr 0.8 9.3 f 0.6

*VR DR 21.4 -t 1.6 11.3 f 0.5 VR 12.4 f 1.3 9.3 + 2.4

Numerous studies with chick embryo ret-

inal cells have established that adhesion- related phenomena are trypsin sensitive (Moscona, 1974). Table 2 shows that adhesion between dorsal and ventral retinal cells is more trypsin sensitive than adhesion between cells from the same half of retina.

” Incubation was for 30 min at 37°C. ’ SE of quadruplicate determinations.

McQuiddy and Lilien (1971) demonstrated that trypsinization depletes half of

residues in adhesion was investigated by preincubating labeled cells with either C. perfringens neuraminidase or sodium metaperiodate. Mild periodate treatment of animal cells results in specific oxidative cleavage of sialyl residues on the cell surface (Presant and Parker, 1976; Gahmberg

retinal cell sialic acid. The role of sialyl and Andersson, 1977). After either treat-

CAFFERATA, PANOSIAN, AND BORDLEY Embryonic Retinal Cell Adhesive SpecificltJ 113

ment, *DR cells, but not *VR cells, showed reduced adhesive preference (Fig. 4). Adhe- sion between cells from the same half of retina is essentially unaffected by these reagents. The coincidence of these results is no less surprising considering that the conditions of each set of preincubations varied drastically, e.g., pH 7.3 at 0°C for 10 min in

‘VR

I 1 0 005 05 5 5 05 i

SODIUM PERIODATE CONCENTRATION jmM)

FIG. 4. Effect on adhesive preference of preincubating *DR and *VR cells with neuraminidase (A) or metaperiodate (B). Adhesion was measured after a 30. min incubation at 37°C. Each point represents the mean of four determinations; error bars for SE. One unit of neuraminidase causes the release of 1 pmole of sialic acid per minute from bovine submaxillary mucin at 37°C and pH 5; for the dilutions used in this experiment, no detectable hydrolysis ofp-nitrophenyl- /3-o-galactoside or p-nitrophenyl-P-D-N-acetylglucos- aminide occurred after 1 hr at 37°C and pH 4.3. Note: Triangles denote periodate treatments in the presence of 10 m&f a-D-ghCOSe.

the case of periodate and pH 6.0 at 37°C for 30 min in the case of neuraminidase.

Borotritiate labeling. Treating *DR cells with NaBHl does not affect adhesive preference (Fig. 5). However, periodate-induced inactivation of adhesive preference was reversed by subsequent treatment with borohydride. Accordingly, it should be possi- ble to label the cell surface molecules crucial to the reactivation of adhesive preference by reacting periodate-treated cells with borotritiate. This coupled reaction specifically labels the surface sialoglycoproteins of erythrocytes (Liao et al, 1973; Steck and Dawson, 1974; Gahmberg and Anders- son, 1977) and lymphocytes (Gahmberg and Andersson, 1977; Mitchell and Rowers, 1977).

After periodate-borotritiate reaction of intact dorsal and ventral retinal cells, lysates were prepared and analyzed b.v electrophoresis. Figure 6 illustrates the pattern

II- - NoI04 NaIOd

ICI - NaBH, - NaBHd

TREATMENT

FIG. 5. Recovery of *DR cell adhesive preference by reaction of periodate-treated cells with borohydride. *DR cells were treated with 0.5 mM NaIO.$ as indicated. The cells were then washed, resuspended at the same density in normal saline + Hepes containing 0.5 mM NaBH, (as indicated), and incubated for 80 min at 0°C. Afterwards the cells were washed and resuspended in assay medium. Each point represents the mean of four determinations; error bars are for SE.


MWx103

p48- 200=

94-

68-

40.

12 34 56 78 910 FIG. 6. Fluorogram of polyacrylamide slab gel with lysates from NaBTI-labeled retinal cells. Samples were

prepared from 2 X lo7 cells. After electrophoresis each lane in the slab stained to a similar intensity with Coomassie brillant blue (except lane 3). Migration is from top to bottom. Molecular weights were estimated from the migration of the spectrins, myosin, phosphorylase A, BSA, and horesradish peroxidase in the same gel. Odd- and even-numbered lanes represent lysates of dorsal and ventral cells, respectively. Periodate concentration: (1, 2) none; (3, 4) 5 mm, (5, 6) 0.5 mM, (7, 8) 0.05 mM, (9, 10) 0.005 mM.

of radioactive bands in gel fluorograms of these samples. Periodate (0.05 m&f) induced the appearance of two main bands at 90,000 daltons (GP90) and 40,000 daltons (GP40) (compare lanes 1 and 2 with lanes 7 and 8). Although reproducible, these molecular weights represent estimates since the bands contain carbohydrate (Segrest et

al., 1971). A third main band appeared at the ion front and did not require periodate to be labeled. This band was not subfrac- tionated on 15% acrylamide gels and probably represents borotritiate reduction of C=C bonds in membrane lipids.

In addition, at least 22 other bands were labeled at higher periodate concentrations

CAFFERATA, PANOSIAN, AND BORDLEY Embryonic Retinal Cell Adhesioe Specificitv 115

(0.5 or 5 n&f). However the reactivity and/or accessibility of GP90 and GP40 to periodate-borotritiate parallel periodate’s effect on the adhesive preference of *DR cells (Fig. 7). This comparison suggests that it is necessary to modify only a few different surface constituents to inactivate adhesive preference. Ventral retinal cells do not appear to be deficient in these constituents.

Treatment of cells with either neuraminidase or periodate probably affects similar surface constituents, since incubating periodate-borotritiate-labeled cells with neuraminidase released 80% of the tritium not attributable to the borotritiate treatment alone (data not shown). Digesting labeled cells with 0.1 N H,SO, at 80°C for 1 hr also released 80% of this label, suggesting that the tritium was incorporated in sialic acids.

DISCUSSION

The most exciting possibility raised by these results is that adhesive preference between cells, dispersed mechanically from dorsal and ventral half-retinas, depends on surface carbohydrates, possibly sialic acids. Support for this statement is based on the

0 o.o& 065

SODIUM PERIODATE CONCENTRATION (mM)

FIG. 7. The effect of periodate on the borotritiate labeling of GP90 and GP40 is correlated with the loss of adhesive preference. The adhesive response of *DR cells to periodate is replotted from Fig. 3B. Levels of GP90 and GP40 are compared as the relative areas of their respective peaks in the densitometry traces of Fig. 5 (not shown). Filled symbols, dorsal cells; open symbols, ventral cells.

finding that this pattern of intercellular adhesion is markedly reduced following previous treatment of dorsal retinal cells with neuraminidase or periodate. Since these reagents fail to reduce adhesion of ventral retinal cells to dorsal retinal cells or between cells from the same half-retina, it seems reasonable to assume that these interactions do not involve such carbohydrate moieties.

These results and the trypsin sensitivity of complementary adhesive preference in- dicate the importance of protein-carbohydrate interactions in this adhesive pattern Marchase (1977) reached a similar conclusion after studying the adhesion of dorsal and ventral retinal cells for complementary halves of tectum. In that assay, the activity of ventral retinal cells is trypsin sensitive and that of dorsal retinal cells is sensitive to P-N-acetylhexosaminidase but not trypsin or neuraminidase. The latter discrepancy with our results has several explanations. First, the cells used by Mar- chase (1977) were prepared by trypsinization and, therefore, probably lack numerous surface glycoproteins. Nevertheless, the dorsal retinal cells maintain adhesive preference with other glycoconjugates that are resistant to proteases (e.g., gangliosides). Second, the type of glycoconjugates used by dorsal retinal cells could differ depending on whether adhesion is measured to cell layers or the acellular surface of tecta. Third, identical populations of dorsal retinal cells might not be active in each assay. According to Marchase (1977), less than I’P of the incubated cells formed stable attach- ments to tecta. Under our conditions, adhesive efficiency exceeds that figure by more than an order of magnitude. Dorsal retinal cells treated with neuraminidase or periodate do retain a slight preference for layers of ventral retinal cells. This residual preference might be the response of a minor fraction of cells which, nevertheless, com- prise the majority of cells adherent to tecta. At this time, it is impossible to choose between these alternatives.


Tyler (1942) snd Weiss (1947) have sug- gested that specific cell-cell associations involve the interaction of complementary molecules similar to that which occurs in the reaction of antigens and antibodies. This hypothesis is consistent with two of our present observations: (i) Adhesive preference is detectable at O’C; and (ii) periodate’s effect on adhesive preference is pro- duced at 0°C. These results confirm those of Curtis et al. (1975), that embryonic retinal cells, prepared without trypsinization, exhibit favorable adhesive rates at low tem- perature.

How might carbohydrate moieties on dorsal retinal cells mediate adhesive preference? Our experiments cannot distin- guish between a direct or indirect role for sugars, i.e., whether these residues interact with adhesive receptors on ventral cells or influence the affinity of adhesive sites on dorsal cells. Recent experiments show that the presence of 5 mM N-acetylneuraminic acid fails to block adhesive preference (data not shown). The failure of monosaccharide to compete with cell surface residues does not preclude a direct role of this or other sugars. Recognition might be more compli- cated than the interaction between a simple sugar and a complementary site on a cell. In the interaction of myxoviruses and par- amyxoviruses with sialic acids, neither the free sugar, the sialoglycopeptides, nor the gangliosides show receptor activity, whereas native sialoglycoproteins or lipo- somes containing gangliosides do (Gotts- chalk and Fazekas de St. Groth, 1960; Hay- wood, 1974). It would be of interest to examine the possibility that complementary adhesive preference involves recognition of a critical array of carbohydrates. Several surface sialoglycoproteins have been iden- tified which could function in this manner.

From the preceding discussion it is not obvious why borohydride reactivates periodate-induced loss of adhesive preference. Bone marrow cells, subjected to these treatments, regain adhesive properties necessary for implantation and hematopoiesis in the

spleen (Tonelli and Meints, 1978). In both cases, coupled periodate-borotritiate reac- tions yield a product degraded by neuraminidase. This is consistent with periodate oxidation of sialic acids at exocyclic carbons generating carbon-7 aldehydes (Van Lenten and Ashwell, 1971). Borohydride would reduce these aldehydes to the corresponding hydroxy analogs. Depending on whether such carbohydrates act directly or indi- rectly in adhesive preference, this reaction could be interpreted as regenerating receptor-like ligands or blocking the Schiff s base reactivity of aldehydes on the membrane. The latter has been emphasized to explain the inhibition by reducing agents of periodate’s mitogenic effect on lymphocytes (Novogrodsky and Katchalski, 1972; Pre- sant and Parker, 1976).

Our data on the response of adhesive preference to eye inversion are consistent with other studies of axiality in the developing chick retina. Attempts to reorient the optic fissure (Goldberg, 1976) or reduce the retinotectal projection (Crossland et al., 1974) by surgical manipulation of the eye are successful if conducted after stage 12 of development. Each of these expressions of differentiation appears to respond to an axial system which fixes its reference points within the eye during stage 12. The question of whether stage 12 is the critical pe- riod in the determination of complementary adhesive preference remains to be studied. Answering that question could pro- vide insight into the embryonic regulation of molecules specifying cell adhesion.

We thank Drs. P. R. Gross, A. Haywood, J. Holt- freter, and G. Marinetti for helpful discussions during the course of this work. We also thank Dr. G. Baum, Corning Research and Development Laboratories, for advice on the reactivity of silane derivatives and Mary Drislane for assistance in beginning these experiments. This work was supported by Grant EY-01529 from the National Institutes of Health to R. C. and a Rochester Plan Undergraduate Fellowship to J. P.

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Developmental and biochemical studies of adhesive specificity among embryonic retinal cells

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