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Studies of the Developing Chick Retina Using Monoclonal Antibody 8A2 That Recognizes a Novel Set of Gangliosides *Department

of Neurobiology. and

Anatomy and Cell Science, University of Pittsburgh School of’ Medicine, Pittsburgh, Pennsylvania of Cell Biology and Anatomy, University of Miami School of Medicine, P.O. Box 016960, Rosensteil Medical Sciences Building, 1600 Northwest 10th Avenue, Miami, Florida 33101

15261;

fDepartme&

Accepted

January

2X. 1991

A monoclonal antibody, Mab 8A2, that recognizes a novel set of gangliosides was produced by immunizing a mouse with Embryonic Day 14 chick optic nerve. Immunohistochemical studies of the developing chick retina revealed a complex pattern of Mab 8A2 immunoreactivity. Initially, staining is concentrated in the optic fiber layer in the central retina. Later in development, the most intense staining is seen at the periphery of the retina and 8A2 immunoreactivity appears in other retina layers. In the adult retina, 8A2 immunoreactivity is lost from the optic fiber layer but persists in the inner plexiform layer, inner nuclear layer, and outer plexiform layer. Cell culture experiments showed intense staining of neurites from retinal ganglion cells but no staining of Muller cells. Biochemical characterization of the epitope recognized by Mab 8A2 suggests that it includes a 9-0-acetyl group that is present on five different gangliosides. The 8A2 immunoreactive gangliosides are distinct from and have slower mobilities on thin-layer chromatographs than those recognized by Mab Dl.l which recognizes I)-0-acetyl GD3. ZI 1991 Academic press. I~C.

INTRODUCTION

Gangliosides are a complex group of sialic acid containing glycosphingolipids that occur in high concentration in the central nervous system. While there is no definitive evidence of their specific functions, a number of potential activities have been ascribed to them. Since gangliosides comprise a significant proportion of the total glycoconjugates that exist on the surface of neuronal cells, they have been assumed to be involved in many activities in which the cell surface plays a role. Their localization in regions of cell-cell contacts implicates them as potential mediators of cell-cell recognition (Hansson et ah, 1977). Gangliosides support neural retinal cell adhesion in vitro (Blackburn et al., 1986) as well as promote neuritogenesis in both primary neuronal cultures (Roisen et al., 1981) and established neuronal cell lines (Byrne et al., 1983; Leon et ah, 1982; Roisen et ah, 1981). Gangliosides show qualitative and quantitative changes during development (Dreyfus et al., 1975; Irwin et al., 1985; Landa and Moscona, 1985; Mintz et al., 1981; Panzetta et ab, 1980), further suggesting their involvement in cell surface activities which function during development.

’ Current address: NIH/NINDS, Building 36 Room 2829,900O Rockville Pike, Bethesda, MD 20892. ‘To whom correspondence and reprint requests should be addressed at Department of Neurosciences, Case Western Reserve University, Cleveland, OH 44106. 0012-1606/91 Copyright All rights

$3.00

c‘ 1991 by Academic Press, Inc. of reproduction in any form reserved.

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Several investigators studying the development of the vertebrate nervous system have produced monoclonal antibodies against gangliosides. (Constantine-Paton et al., 1986; Eisenbarth et al., 1979; Grunwald et al., 1985; Levine ef ab, 1984; Rosner et al, 1985). These antibodies have exhibited spatially and temporally diverse immunohistochemical staining patterns that may be attributed to the diversity of glycoconjugate expression at different stages of development. Characterizing the distribution and appearance of such cell surface molecules will aid in understanding more fully their role in neuromorphogenesis. In this report, we describe a novel monoclonal antibody, 8A2, which binds to an epitope expressed on a group of gangliosides that show interesting developmental regulation. The antigens are strongly expressed on growing axons in the chicken central nervous system, and are specific for chick neurons in vitro. By virtue of their localization, the 8A2 antigens have proven very useful for immunohistochemical staining of retinal axons in tissue culture. MATERIALS

AND

METHODS

Embryos. Fertilized chicken eggs, (White Leghorn) obtained from Sachs and Sons Poultry Farm (Evans City, PA) were incubated in a forced-draft incubator at 39°C. Embryos were staged according to Hamburger and Hamilton (Hamburger and Hamilton, 1951). Immunixations and fusion. Optic nerves were dissected from seven dozen Embryonic Day 14 (E14) chick

DRAZBA,PIERCE,ANDLEMMON

embryos and homogenized in 5 ml of Dulbecco’s phosphate-buffered saline (PBS).3 Mice were immunized with 0.1 ml of homogenate at 4-week intervals followed by a series of three iv injections on successive days and removal of the spleen on the fourth day. Hybridomas were prepared with NS-1 myelomas using standard protocols (De St. Groth and Scheidegger, 1980). An immunohistochemical screening procedure was used to select for monoclonal antibodies specific for ganglion cells in the chick retina. After screening, positive wells were cloned and recloned by limiting dilution until over 90% of the clones were positive. Cell stocks were then frozen and stored in liquid nitrogen. Ganglioside cha,racterixation. Embryonic Day lo-13 chick retinas, tecta, and brain were subjected to Folch’s partition method (Folch et al., 1957) and the ganglioside fraction was resuspended in distilled water. The extracts were desalted by Sep-Pak Cl8 cartridge chromatography (Kundu and Suzuki, 1981), then diluted lo-fold with chloroform:methanol (C:M) (2:1), and spotted on high-performance thin-layer chromatography (HPTLC) plates. A solvent of n-propanol:0.25% KC1 (aqueous) (7:3) was used to separate the gangliosides, after which the plate was immunostained using the 8A2 monoclonal antibody and a Vectastain ABC, anti-mouse IgM kit (Buehler and Macher, 1986). Mab D1.l, which was kindly provided by Joel Levine, was also used to stain plates to allow a direct comparison of the binding specificities of these two antibodies. GMl, GDla, and GTlb (Supelco, Inc.) were used as standards and were visualized using iodine vapors. For some experiments, gangliosides that bound Mab 8A2 were purified using a DEAE-Sephadex column where the extract was applied in C:M:W (30:60:8) and step-eluted with ammonium acetate in methanol (Ledeen and Yu, 1982). About 385 pg of sialic acid equivalents, quantitated by the method of (Powell and Hart (1986), was dried from 95% ethanol onto individual wells of a 96-well microtiter plate and an ELISA was performed using 8A2 culture supernatant and the Vectastain ABC kit. Some aliquots were treated with either mild base (0.1 N NaOH, 3 hr, 37°C) or mild periodate treatment (4 mM periodate, lo’, 4°C) (Blum and Barnstable, 1987). Immunohistoch~ernistry. Frozen sections: In order to characterize the expression of the 8A2 antigens during development, retinae from embryos age E3 through adult were fixed for 3 hr at 4°C with 1% glutaraldehyde in 0.1 Mphosphate buffer, cryoprotected in 20% sucrose in PBS at 4°C for 24-48 hr depending on age, embedded 3 Abbreviations used: avidin-biotin-horseradish peroxidase complex, ABC; fetal calf serum, FCS: Dulhecco’s phosphate-buffered saline, PBS; high-performance thin-layer chromatography, HPTLC; horse serum, HS; Mah, monoclonal antibody; penicillin-streptomycin, PS.

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in OCT mounting medium (Tissue-Tek, Miles Laboratories Inc., Naperville, IL), and frozen in liquid nitrogen. Cryostat sections were cut at lo-15 pm, mounted on chromium potassium sulfate-gelatin-coated microscope slides and allowed to dry. The following procedures were conducted at room temperature. After incubation with hybridoma supernatant for 1 hr, sections were washed three times with PBS and incubated with biotinylated goat anti-mouse IgM (Vectastain, Vector Laboratories; Burlingame, CA) in PBS/lo% horse serum (HS) for 1 hr. Following three PBS washes, sections were incubated with avidin-biotin-horseradish peroxidase complex (ABC) (Vectastain, Vector Laboratories) for 1 hr. After three final washes in PBS, the sections were reacted with diaminobenzidine/cobalt chloride/nickel ammonium sulfate solutions (Adams, 1981). To determine specificity and cross-reactivity, immunohistochemistry with the 8A2 antibody was performed on chick optic tectum, cerebellum, kidney, liver, intestine, skeletal muscle, and cardiac muscle, as well as El5 rat retina and brain. For both light and electron microscopic studies (see below), controls were performed that included incubating sections without the primary antibody, but with secondary antibodies to determine if secondary antibodies bound nonspecifically to the tissue. We found no binding of HRP-labeled antibodies or ABC complex on sections incubated only with these reagents. Electron microscopic immunohistochemistry. Eyes taken from E6 and El0 chick embryos were fixed by immersion for 5 min at 4°C in 4% paraformaldehyde/ O.Ol%> glutaraldehyde in 0.1 MPipes buffer, pH 7.4. Subsequently the retinae were removed, washed briefly in PBS, and then whole mounted onto concanavalin Acoated nitrocellulose paper. The mounted retinae were immersed in the same fix for 4 hr at 4°C washed in PBS, and cut into strips. The strips were incubated at 4°C for 30 min in 100% horse serum followed in some cases by the same incubation in PBS/lo% HS/O.Ol% Triton X-100. Strips were then incubated in either Mab 8A2 (intact IgMs or bivalent fragments (Matthew and Reichardt, 1982)) or Mab 8D9 (an IgG which binds to the axonal cell adhesion molecule Ll (Lemmon and McLoon, 1986)) overnight at 4°C on a rocker. Next, the sections were washed four times with PBS/lo% HS over a 1-hr period before incubation with a biotin-labeled secondary antibody for 1 hr at 4°C. Sections were again washed four times and then incubated with avidin-biotin-HRP complex (Vectastain, Vector Laboratories). After a final series of washes, the sections were processed with an intensified diaminobenzidine procedure (Adams, 1981). The sections were then postfixed in 2% glutaraldehyde/ 0.1 M phosphate buffer15% sucrose, washed four times over a 1-hr period with 0.1 M phosphate buffer/5% sucrose, and then postfixed in 1% osmium in 0.1 M phos-

156

FIG. retina amount retina,

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BIOLOGY

1. Immunostaining of total ganglioside fraction from ElO-13 and tecta, as described under Materials and Methods. The of material spotted represents that from 20 retina, lane A; 40 lane B; 20 tecta, lane C; 40 tecta, lane D.

phate buffer with 1 mg/ml ferrocyanide for 45 min on ice. The strips were then dehydrated and embedded in Epon 812/araldite. Gold-colored sections were cut and examined using a JEOL 100 CX EM. Tissue culture. Preparation of retinal cell cultures: Confluent monolayers of chick retina Muller cells were prepared by culturing dissociated El3 retinae as follows: neural retinae were isolated and incubated for 30 min at 37°C in 0.125% trypsin in Hank’s Ca2+ Mg”-free saline solution. Fetal calf serum (FCS) was then added to at least 20%) and DNase was added to 0.05% prior to final trituration of the tissue. The cells were pelleted at 8OOXg for 5 min and then resuspended in DMEM/lO% FCWpen-strep (GIBCO/BRL Laboratory, Gaithersburg, MD). This procedure produced single cells with a viability of greater than 95% based on trypan blue exclusion. The cells were plated at a density of 1 X lo6 cells/cm’ in 35-mm plastic tissue culture dishes (Falcon Labware) in DMEM/lO% FCS containing penicillinstreptomycin (PS) (GIBCO/BRL Laboratory) and were incubated at 37°C in 7% C02, 93% air, for 3 days. Neurons were removed by treating the cultures with the 8A2 monoclonal antibody in the presence of 4% rabbit complement. Following two 48hr pulses with 20 and 40 pg/ ml BUDR, respectively, in DMEM/lO% FCS/PS, strips of E6 retina were placed on the Muller cells according to the method described in Drazba and Lemmon (1990). Axons from the retinal ganglion cells were allowed to grow out from the explants. Live cultures were incubated with monoclonal antibody 8A2 for 60 min at 37°C and then fixed with 1% glutaraldehyde in 0.1 M phosphate buffer for 15 min at room temperature. After washing with PBS and blocking with 100% horse serum for 10 min, cultures were processed with Vectastain and DAB as described above for frozen sections.

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retina, while being differentially distributed across the remainder of the retina. Preliminary screening of other chick tissues indicated that antibody 8A2 recognized only those of neural origin. Strong staining of developing optic tectum and cerebellum was observed, but no stain was found in kidney, liver, intestine, skeletal, or cardiac muscle. The 8A2 antigens were strongly expressed in El5 rat brain, but were completely absent in the rat retina (data not shown). Biochemical characterization of the 8A2 antigens. Preliminary attempts to characterize the 8A2 antigen indicated that it was completely resistant to a variety of proteases, but was sensitive to neuraminidase, suggesting the antigen might be a ganglioside (data not shown). Folch’s partition method (Folch et al., 1957) was used to obtain crude retinal and brain gangliosides. Thin-layer chromatography of the desalted gangliosides, followed by immunostaining (Buehler and Macher, 1986), revealed that Mab 8A2 bound to at least five bands present in both ganglioside extracts (Fig. 1). Comparison of the binding pattern of Mab 8A2 with that of Mab D1.l demonstrated clearly that these two antibodies do not recognize the same set of gangliosides (Fig. 2). ELISA determinations revealed that all 8A2 immunoreactivity bound and was eluted from DEAE-Sephadex by ammonium acetate (data not shown). Figure 3 shows that mild base treatment completely eliminated 8A2 binding to the ganglioside fraction while mild periodate treatment did not. After HPTLC, immunostaining confirmed that all reactive species lost binding activity after base treatment but were unaffected by mild periodate treatment. Expression of antigens 8A2 during development. At E3 strong staining of the central retina was present, apparently of axons from the first ganglion cells. Strong staining was also seen in the ventral portion of the developing optic stalk, presumably on axons as they course along their path to the tectum. Intense staining was A

B

C

RESULTS

Initial characterization of antibody 8A2 revealed intense staining of the optic fiber layer of developing chick

FIG. 2. Comparison of Mab XA2 and Mab D1.l immunoreactivity of extracts from El4 chick retina. Lane A, iodine visualization of total extract; lane 8, Dl.1 immunoreactivity; lane C, 8A2 immunoreactivity.

Xtl,

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157

G(rl/~~/i/KIdf's

A UNTREATED -

BASE

0.8 -

00 ANTIBODY

FIG. 3. ELISAs of ganglioside extracts treatment eliminated 8A2 binding while

ANTIBODY

DILUTION

showing periodate

effect of base treatment treatment had no effect.

seen in the region of the optic chiasm and throughout the diencephalon (Fig. 4A). At E6 strong staining continued in the developing optic tract and optic nerve. Diffuse stain could be seen throughout the stratified neuroepithelium of the central retina with stronger staining visible adjacent to the inner limiting membrane. At this stage the stain was strongest in the central retina (Fig. 4B). As the retina matures, the 8A2 antigens are progressively expressed on cells more peripherally. At El1 stain could be seen throughout the entire retina with a concentration in the optic fiber layer, optic nerve, and newly formed inner plexiform layer of the central retina (Fig. 4C). By El5 the expression of antigens was more pronounced peripherally than centrally. Although the optic nerve and optic fiber layer continued to exhibit strong staining throughout, the inner and outer plexiform layers expressed more immunoreactivity peripherally (Fig. 4D). In addition, the differentiating marginal retina within 200 pm of the ora serrata was very intensely labeled (Fig. 4D). At El8 8A2 immunoreactivity was expressed in the inner and outer plexiform layers and also began to appear in the inner nuclear layer. Strong expression of immunoreactivity continued in the optic nerve and optic fiber layer. Peripheral retina continued to stain more intensely than central retina. Immunohistochemistry of the adult retina demonstrated significant differences from the developmental period (Fig. 5). In the adult, the optic nerve and optic fiber layer no longer expressed the 8A2 immunoreactivity. The staining pattern appeared similar throughout the retina with strong staining of the inner plexiform, inner nuclear, outer plexiform layers, and the outer lim-

(A)

and periodate

treatment

DILUTION

(B) on 8AZ immunoreactivity.

Base

iting membrane. The 8A2 immunoreactivity was not expressed on the inner or outer segments of photoreceptor cells at any age (Table 1). A summary of the expression of 8A2 immunoreactivity at different developmental stages is included in Table 1. Electron microscopic loculixation of the 8A2 antigem. Immunohistochemical studies at El0 with proteasetreated 8A2 antibody indicated that the antigens were not present on all retinal cell axons (Fig. 6B). Some axons were intensely stained while nearby axons were weakly stained or not stained. As a control, antibodies to the axonal cell adhesion molecule Ll (Lemmon and McLoon, 1986), which is present on the axons of all retinal ganglion cells, were used to stain adjacent pieces of retina. As expected, all retinal cell axons appeared uniformly stained (Fig. 6A). Thus, the nonuniform staining of the El0 axons by Mab 8A2 was not due to penetration problems. When E6 retina was stained with Mab 8A2 fragments, all axons uniformly expressed 8A2 immunoreactivity (Figs. 6C and 6D). These results are consistent with a decreased expression of 8A2 antigens by axons of retinal ganglion cells with increasing age. Expression of8A.2 immunoreactivity bg cultured cells. Retinae from El3 chick embryos were dissociated into single cells and cultured in plastic tissue culture dishes for 3 days. The cultures consisted of large flat cells spread over much of the dish, with clumps of neurons adhering to their upper surfaces. When cultures were treated with the 8A2 antibody in the presence of rabbit complement, neurons on the culture surface were destroyed, while the underlying Muller cell monolayer was left intact. When E6 retinal strips were placed on these monolayers, allowed to extend processes, and stained

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FIG. 4. Expression of 8A2 immunoreactivity in chick retina at early developmental stages. At E3 (A) and E6 (B) strong staining is seen in the central retina, optic fiber layer, ventral optic stalk, optic chiasm, and diencephalon. At El1 (C) and El5 (D) 8A2 staining is present throughout the retina and is concentrated in the optic fiber layer and inner plexiform layer. The peripheral retina stains more intensely than the central retina. Scale bars: A-C, 350 Km; D, 1 mm.

with Mab 8A2, the processes from the E6 strips were intensely stained while underlying Muller cells remained unstained (Fig. 7’). DISCUSSION

In the present study, we describe the production of monoclonal antibody 8A2 and the preliminary characterization of its antigens in the developing chick visual system. Analysis of material prepared by Folch’s partition procedure revealed no 8A2 immunoreactivity in the neutral glycolipid fraction, while all 8A2 immunoreactive material in the ganglioside fraction was eluted from a DEAE column by ammonium acetate. The sensitivity of Mab 8A2 binding to mild base treatment and resistance to mild periodate treatment suggest that the 8A2

epitope includes a 9-0-acetyl-sialic acid (Cheresh et al., 1984) or possibly an internal lactone. These results suggested a relationship between Mab 8A2 and Mabs Dl.l/ JONES which recognize 9-0-acetyl-GD3. A direct comparison of Mab 8A2 and Mab D1.l on immunoblots revealed that they recognize different sets of gangliosides. This result is consistent with the differences in their histochemical staining patterns of nervous tissue For example, the 8A2 antibody stains Embryonic Day 15 rat brain, but does not recognize Embryonic Day 15 rat retina, while JONES stains both. One intriguing aspect of immunoblots of gangliosides from retina and tectum is that the mobilities and relative amounts of 8A2-positive bands are very similar in the two tissues. This result contrasts with comparisons of the major forms of gangliosides, most of which show significant differences be-

DRAZBA,

PIERCE,

AND LEMMON

X.42

(;a r~qliosidw

159

C

FIG. 5. Expression of 8A2 immunoreactivity in the adult chicken retina. Dorsal (A and B) and central (C and D) retina show similar patterns of staining in the inner plexiform (IPL), inner nuclear layer (INL), and outer plexiform layer (OPL). The optic fiber layer (OFL) and the optic nerve head in E no longer express SA2 immunoreactivity. Scale bars: A, C, and E, I mm, B and D, 50 pm.

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TABLE 1 SUMMARY OF Mab 8A2 IMMUNOREACTIVITY OF DIFFERENT RETINAL LAYERS DURING DEVELOPMENT E6 Layer

E3

c

Optic fiber Inner plexiform Inner nuclear Outer plexiform Outer nuclear Photoreceptor Optic nerve

++

++ + +

t+

++

El1

E8 P

c

P

El5

El8

ADULT

C

P

C

P

c

P

C

P

++ + +

++ ++ +

+ + i

++ + +

++ ++ + +

++ + + +

++ + t-+ +

++ ++ ++

++ ++ ++

++

++

-t t

++

lvotu. C, central; P, peripheral. In developing retina the XA2 antigen is present in the OFL, ON, and to a lesser degree in the IPL. In the mature retina the 8A2 antigen is present in the IPL, INL, and OPL. In the developing retina the 8A2 antigen appears to be concentrated on the cell surface, while in the mature retina it appears to be concentrated intracellnlarly in ganglion cells and cells of the INL.

tween the retina and brain (Dreyfus et al., 1975). These results also imply that the activities of specific glycosyltransferases and other enzymes responsible for the biosynthesis of the 8A2 epitope are similarly expressed in both retina and tectum. Since our immunohistochemical studies show that axons are the predominant site of 8A2 immunoreactivity at ElO-14, perhaps axons in different parts of the CNS express a similar pattern of 8A2 immunoreactive gangliosides. At early embryonic stages of development, 8A2 immunoreactivity is observed in central regions of the retina during genesis of the first postmitotic, differentiated cells (Rager, 1976). As early retinal development continues, antigen expression follows the pattern of maturation, with the most intense staining found predominantly in those regions of the retina that are postmitotic. At about E9, the cells in the central retina undergo their last division and mitosis is then confined to the margin of the tissue (Coulombre, 1955). During this time some cells in the central retina ceased to express the antigens, while cells of the peripheral retina, which was still in the process of proliferation, continued to stain intensely. On the basis of these observations, a correlation between antigen expression and the postmitotic age of individual cells can be suggested. However, during late stages of embryonic development when the neural retina possessesa definitive complement of postmitotic cells, different patterns of 8A2 expression are exhibited in the retina with the persistence of intense staining in the periphery. In the adult, 8A2 immunoreactivity is significantly different from that in earlier developmental stages. The most notable change in the adult retina was the loss of 8A2 immunoreactivity in the optic nerve and optic fiber layer, which were areas that consistently expressed immunoreactivity intensely throughout development. This loss suggests that differential expression of the antigens is related to factors

other than the age of an individual cell. The expression may be related to retinal position, to the state of differentiation of a particular cell, or to a role yet to be determined in cell-cell interactions. Intense binding of the 8A2 antibody to the optic fiber layer and optic nerve led to a detailed study of expression of the antigens on ganglion cells and their axons. Electron microscopic studies indicated that at E6, all of the axons in the optic fiber layer expressed immunoreactivity, but by ElO, only about half continued to do so. While early axons could be stained, many somas in the ganglion cell layer showed little or no staining. Although the 8A2 antibody is an IgM, less than optimal penetration is not likely the reason for the selectivity since the antibody underwent protease treatment to reduce its size before usage. The treatment produced fragments of approximately 150,000kDa, which are assumed to be bivalent (Matthew and Reichardt, 1982). Also, axons of companion sections, stained with intact Mab 8D9 IgGs which bind to the Ll cell adhesion molecule present on all retinal axons, are uniformly labeled, suggesting that molecules at least as large as an IgG were able to penetrate the tissue. The ability of intact IgMs to label all of the axons in the E6 retina also indicates that penetration is not a significant problem. The selective pattern of 8A2 expression observed in vivo is conserved when cultures of freshly dissociated embryonic retinal cells are stained with the antibody. Most neurons stain very intensely while some are devoid of stain. Muller cells do not bind the antibody. This finding is consistent with the result obtained when dissociated cultures are treated with rabbit complement in the presence of the 8A2 antibody. While Muller cells remain a nonaffected monolayer, the neurons are lysed and washed away. Similarly, when E6 retinal explants are cultured on Muller cell monolayers and stained with the 8A2 antibody, ganglion cell processes which have

DRAZBA,

FIG. 6. Electron immunoreactive. immunoreactive.

PIERCE,

AND

LEMMON

X42

Gtr r~q1iosirlr.s

microscopic immunohistochemistry of E6 and El0 retina. (A) Staining with Mab 8DS of El0 retina shows all axons (B) Staining of sections from the same retina as in A with Mab 8A2 shows that only some axons, usually in clusters, (C and D) Staining of E6 retina with Mab 8A2 reveals that all axons are intensely labeled. Scale bars = 1 pm.

are are

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FIG. 7. 8A2 staining image

showing

intense

of retinal staining

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explants on monolayer of Muller cells. (A) Phase-contrast of neurites from explant and no staining of glia. Scale bar

grown out stain intensely while underlying Muller cells remain unstained. The biochemical experiments showing that Mab 8A2 recognizes at least five different ganglioside species may help to explain the varied patterns of immunohistochemical staining seen at different locations in the retina at different developmental stages. Perhaps different 8A2 reactive gangliosides are expressed at different times or in different cellular locations, for example axons versus dendrites. Abundant work has been done describing both the quantitative and the qualitative changes in the ganglioside profile of the developing nervous system in many species. In the chicken these changes are most pronounced during the prehatching period with increasing amounts of more complex gangliosides appearing at later stages of development in vivo (Dreyfus et al, 1975; Fredman et ab, 1984). The appearance of the 8A2 antigens coincides with the appearance of differentiating cells in the retina. The first appearance of the 8A2 antigens in the chick retina occurs when retinal ganglion cell axons first emerge. Strong staining is present on the retinal ganglion cell axons as they grow along the path that they take to reach their targets as well as in the regions that they invade. During later development strong 8A2 immunoreactivity was consistently observed at the extreme periphery of the retina where cell proliferation occurs. Although many antiganglioside monoclonal antibodies have been described in recent years, only two, the JONES antibody and Mab D1.l, both of which bind to 9-0-acetyl-GD3 (Cheresh et al., 1984; Mendez-Otero et al., 1988), have been found with a pronounced topographically varied staining pattern from dorsal to ventral retina (Constantine-Paton et

image showing = 100 pm.

monolayer

of glia.

(B) Bright-field

al., 1986). In unpublished experiments on E6, E8, and El3 retinas, we found no evidence indicating that the 8A2 gangliosides are present in a dorsal-ventral gradient. Although the functional significance of individual gangliosides in nervous tissue is not yet understood, gangliosides in general are useful biochemical indicators of successive periods of central nervous system development and maturation. The ability of the 8A2 monoclonal antibody to recognize a specific type of ganglioside will further aid investigations of their role in neural development. In addition, the observation that the 8A2 Mab inhibits neurite outgrowth (Pierce et aZ., 1988; Sloan et al., 1989) suggests that 8A2 gangliosides may function in important cell-cell and cell-substrate interactions. Recently, a 53-kDa lectin from vertebrate tissue has been purified and characterized which binds specifically Oacetylated sialic acid residues (Ahmed and Gabius, 1989). It would be interesting to determine if this or similar lectins are present in retina or tecta during neural morphogenesis and whether their location could suggest a function in cell migration or recognition. We thank Joel Levine for providing Mab D1.1. We also acknowledge the excellent technical assistance of U. Reuter-Carlson and T. Massella who performed the EM-immunohistochemical studies and Nancy Cochran and Elliot Ramberg who conducted the ganglioside analysis. This work was supported by Grant EY-5285 to V. Lemmon and Grant EY-6968 to M. Pierce.

Note udded in prooj: Monoclonal antibody 8A2 is available from the Developmental Studies Hybridoma Bank, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205.

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