*Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, and tLaboratory of Biochemical Pharmacology, National Institute of. Arthritis ...
Proc. Nail. Acad. Sci. USA Vol. 82, pp. 4008-4012, June 1985 Biochemistry
Monoclonal antibody 18B8 detects gangliosides associated with neuronal differentiation and synapse formation (hybridoma/ceil surface/retina development/synapse/neuronal differentiation)
GERALD B. GRUNWALD*, PAM FREDMANt*, JOHN L. MAGNANIt, DAVID TRISLER*, VICTOR GINSBURGt, AND MARSHALL NIRENBERG* *Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, and tLaboratory of Biochemical Pharmacology, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20205
Contributed by Marshall Nirenberg, March 22, 1985
Mouse monoclonal antibody 18B8 detects ABSTRACT developmentally regulated antigens in chicken retina and brain. The antigens detected by immunofluorescence appear initially on cell bodies in retinas of 6-13 day embryos. In older embryos during synapse formation and in adults, the antigen is localized in discrete laminae within the inner synaptic layer of retina and also is present in the outer synaptic layer and the outer segments of photoreceptor cells. The antigens from retina and brain were purified partially and were shown to be gangliosides of unknown structure that contain at least two sialic acid residues. Gangliosides that are recognized by antibody 18B8 change both qualitatively and quantitatively during neuronal development. These changes were correlated with the spatial and temporal changes in antigen expression detected histochemically.
The mechanisms involved in neuronal differentiation and the formation of appropriate synapses during neural development are poorly understood. Although progress has been made in the identification of genes that are expressed in a neuron-specific fashion (1-4), little is known regarding how this genetic information is utilized to regulate cell interactions during neural development. One point of control may lie in cell-surface molecules whose structure and organization change during the course of development (5). Such changes may help establish order in the developing nervous system. The retina provides an excellent system for the study of such processes because the neuronal cell bodies and synaptic connections are organized in different layers. Both cellsurface gangliosides and glycoproteins change during retinal development (6-8), although the relationship of these changes to mechanisms of retinal morphogenesis remains unclear. In some cases, antibodies have been used to identify and demonstrate the function of specific surface proteins in the formation of intercellular adhesions between retina cells through calcium-dependent (9) or calcium-independent (10) mechanisms. Recent studies with monoclonal antibodies have demonstrated both a spatial gradient of antigen as well as cell-type-specific antigens in the retina (11-15). In this paper, we describe a monoclonal antibody that binds to some gangliosides in developing retina and brain. Immunohistochemical and biochemical data are presented that show that the species of antigenic gangliosides, their abundance, and distribution in chicken retina change during neuronal development. MATERIALS AND METHODS Production of Hybridomas. Antibody 18B8 is an IgM (K) synthesized by a line of hybridoma cells obtained as de-
scribed (13) by fusion of spleen cells from BALB/c mice that had been immunized with mechanically dissociated 14-day chicken embryo neural retina cells with P3X63 Ag8 mouse myeloma cells (16). Histochemical Procedures. The distribution of antigen in unfixed frozen sections of retina was determined by indirect immunofluorescence. Retinas were dissected in Dulbecco's modified Eagle's minimal essential medium (DME medium) and incubated at 24°C in DME medium containing 10%, 20%, and then 30% sucrose (wt/vol) for 10 min for each solution. Tissues then were embedded in OCT compound (Miles) and frozen on dry ice. Sections 10 gum thick were cut on an IEC cryostat at -20°C, and thaw-mounted on Teflon-grid printed histology slides with 10 viewing areas (Roboz, Washington, DC) that had been cleaned with ethanol. The mounted sections were dried in air for 30 min and stored overnight in a desiccator at 4°C. Sections were hydrated for 20 min in 10%6 normal goat serum and 90% phosphate-buffered saline with Ca2+ and Mg2+ (pH 7.4) (solution A), incubated with antibody (hybridoma-conditioned medium, containing 50 mM Tris'HCl, pH 7.4), for 2 hr at 24°C, washed three times with solution A, and incubated with affinity-purified, fluorescein isothiocyanate-conjugated goat IgG directed against mouse IgG and IgM (Kierkegaard and Perry, Gaithersburg, MD) for 2 hr at 24°C. The sections were washed three times with solution A and mounted in a solution containing 90% glycerol, 100 mM Tris HCl (pH 8.0), and 5.5 mMp-phenylenediamine to retard fading of fluorescence during viewing (17). Sections were examined on a Zeiss Universal microscope equipped for epifluorescence with a BP 485/20 nm excitation filter and a LP 520 nm barrier filter and photographed (Kodak ASA 400 Ektachrome color slide film). The distribution of antigen in unfixed frozen sections of retina cell aggregates was determined as described above. Antigen expression by live retina cells in monolayer culture was determined by indirect immunofluorescence as described above, except that the cells were not incubated in sucrose, embedded, or frozen. Tissue Culture. Retinas from 7-day chicken embryos were dissected in phosphate-buffered saline without Ca2+ or Mg', and single cells were obtained by incubation of the tissue at 37°C for 30 min in phosphate buffered saline without Ca2+ or pancreatic trypsin per ml Mg2e containing 0.5 mg of bovineunits per mg of protein, (three times crystallized, 203 Worthington), followed by dissociation of cells by trituration in Eagle's minimal essential medium (ME medium), containing 10% fetal bovine serum and 0.05 mg of bovine pancreatic DNase I per ml (129,000 units per mg of protein, Calbiochem). Cells then were cultured either as stationary monolayers in 35-mm-diameter tissue culture dishes (Falcon 3001) or as cell aggregates in 35-mm-diameter bacteriological dishes (Falcon 1008) on a rotary shaker platform (70 rpm). All
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tPresent address: Department of Neurochemistry, University of Goteborg, Goteborg, S-414 46, Sweden.
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Biochemistry: Grunwald et al. cultures were maintained in 90% ME medium and 10% fetal bovine serum at 370C in a humidified atmosphere of 5% C02/95% air. One-half of the medium was replaced with fresh medium every third day. Ganglioside Extraction, Purification, and Analysis. Lipids were extracted from chicken brains and retinas with chloroform/methanol/water (4:8:3, vol/vol/vol) as described (18). Total lipid extracts were desalted by dialysis and separated into neutral and acidic fractions by ion-exchange column chromatography on DEAE-Sepharose CL-6B in the acetate form (19). Gangliosides were purified further by silica gel column chromatography (20) and then were fractionated according to the number of sialyl residues by ion-exchange chromatography on Spherosil-DEAE-Dextran (21). Antibody binding to glycolipids and the effect of neuraminidase on antibody binding to gangliosides was measured by solidphase radioimmunoassay as described (22, 23). Ganglioside antigens were detected by immunostaining of thin-layer chromatograms as follows: gangliosides were chromatographed on alumina-backed high-performance thin-layer chromatography plates (Silica gel 60, Merck) in chloroform/ methanol/0.25% KCl in water (5:4:1, vol/vol/vol). Reference gangliosides and gangliosides extracted from tissues, for detection by orcinol reagent, were chromatographed on the same plate as the samples used for autoradiograms so they could be compared. The part of the chromatogram used for autoradiography was soaked for 1 min in 0.1% solution of polyisobutylmethacrylate (Polyscience, Warrington, PA) in hexane. After drying, the plates were sprayed with phosphate buffered saline without Ca2+ or Mg2' and immediately soaked in phosphate buffered saline containing 1% bovine serum albumin (buffer B) for 10 min until the silica gel was wet. The plate was then removed, placed on a slightly smaller glass plate in a Petri dish, overlayed with monoclonal antibody solutions diluted with buffer B to =1 ,g/ml (-60 pl/cm2), and incubated for 3 hr at 4°C. The chromatogram was washed by dipping in four successive changes of cold phosphate buffered saline at 1-min intervals, then placed on a new glass plate in a Petri dish, and immediately overlayed with buffer B (-60 Al/cm2, containing 106 cpm of goat 125I-labeled anti-mouse IgM antibody per ml. After 6 hr at 4°C, the chromatogram was washed as described above in cold phosphate buffered saline, dried, and exposed to x-ray film (XR5, Eastman-Kodak) for 12 hr. Controls without antibody 18B8 were included in each experiment.
RESULTS AND DISCUSSION Expression of Antigen 18B8 During Development. The distribution of molecules recognized by monoclonal antibody 18B8 in unfixed frozen sections of chicken retina at different stages of development was examined by indirect immunofluorescence. At 6 days of embryonic development, the chicken neural retina is a stratified neuroepithelium without synaptic layers. Antibody 18B8 stained intensely some large cells at the vitreal margin of 6-day embryo retina that exhibit ring fluorescence and resemble ganglion neurons, the first neurons to be generated in the retina (24), and also stained some cells at the outer margin of the retina (Fig. 1A). However, most cells in 6-day chicken embryo retina did not bind antibody 18B8 appreciably. Most of the neurons of the retina have been generated by 10 days of embryonic development and the synaptic layers of the retina have appeared; however, synapses that can be identified by ultrastructural criteria are not present (25). Most of the cells in 10-day embryo retina bound antibody 18B8 (Fig. 1B). By 13 days of development, the amount of antigen detected in retina was reduced markedly (Fig. 1C). The amount of antigen detected in some sections of 13-day embryo retina was highest in the inner nuclear layer, especially in the central region where the soma of bipolar neurons, the last neurons in chicken embryo
Proc. Natl. Acad. Sci. USA 82 (1985)
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retina to be generated (24) are located (not shown). By 16 days of development, antigen 18B8 was more abundant in the outer synaptic layer than in other parts of the retina (Fig. 1D). In 18-day chicken embryo retina sections, antigen 18B8 was detected in both the outer and inner synaptic layers (Fig. iE). The retina of the chicken 1 day after hatching is fully functional (26) and resembles the adult retina in histology. Most of the antigen in chicken retina 1 day after hatching was associated with the inner and outer synaptic layers (Fig. IF). In the inner synaptic layer, alternating stained and unstained laminae were seen. Approximately 13 laminae were visible, 7 laminae were stained with antibody 18B8, and 6 had little or no stain. The antigen was distributed in a punctate fashion within each stained lamina. The laminae were not detected when sections were viewed by phase microscopy (not shown). The stained and unstained layers possibly represent layers of synapsing neurites from different types of neurons. The initial stages of formation of laminae with antigen 18B8 were seen in sections of 18-day embryo retina (compare Fig. 1 E and F). Most of the antigen in adult retina also was associated with the inner and outer synaptic layers; however, antigen also was found in the outer segments of photoreceptor cells (Fig. 1G). In oblique sections of adult retina (also newly hatched chicken retina), antigen 18B8 was distributed in the outer synaptic layer in a series of punctate beaded rings (Fig. 1H). Further work is required to determine whether the antigen is associated with photoreceptor synapses or with other synapses in the inner synaptic layer. Control antibody synthesized by parental mouse myeloma P3X63 Ag8 cells did not stain retina sections from adult chickens (Fig. 11) or from embryonic chicken retina (not shown). A section from adult retina stained with hematoxylin and eosin is shown in Fig. 1J to illustrate the architecture of the retina. These results suggest that antibody 18B8 binds to molecules that are expressed by neurons in chicken embryo retina, but not by neuroblasts, and that antigen is associated with the soma of most cells in the retina early in development but becomes progressively restricted during development to some processes in the synaptic layers of the retina and to the outer segments of photoreceptor cells. Expression of Antigen 18B8 by Cultured Cells. Retinas from 7-day chicken embryos were dissociated into single cells, cultured in stationary dishes for 2 weeks, and the living cells were stained with antibody 18B8 to determine the percentage of cells that possess cell-surface antigen. The cells examined for antigen corresponded in chronological age to retina cells of newly hatched chickens. The cultures consisted of confluent monolayers of large flat cells and cells with long processes that resembled neurons that adhered to the upper surfaces of the flat cells (Fig. 2). The cultured cells did not bind control antibody synthesized by P3X63 Ag8 myeloma cells (Fig. 2 A and B). Most flat cells were not stained by antibody 18B8 (Fig. 2 C and D); only a few weakly stained flat cells were found (not shown). However, antibody 18B8 bound to 5% of the cultured cells and most of the cells that bound antibody 18B8 resembled neurons in morphology. Specific immunofluorescence was detected on both cell soma and neurites (Fig. 2 E and F). With sections of retina from newly hatched chickens, antigen 18B8 was detected on neurites but little or none was found on cell bodies. These results show that antigen 18B8 is associated with the external surface of the plasma membrane of some cells and that the distribution of antigens is not restricted only to neurites, as in the intact retina during later stages of development. Further experiments were performed with cells that were dissociated from 7-day chicken embryo retinas and cultured for 2 weeks with shaking to promote the formation of cell aggregates. The distribution of antigen 18B8 in sections of cell aggregates was examined. Many cell bodies and processes were segregated into separate regions within the aggregates, reminiscent of
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FIG. 1. Indirect immunofluorescence analysis of the expression of antigen 18B8 as a function of developmental age. Frozen sections of retina were prepared and stained. All photographs were exposed for the same length of time, so the brightness of the fluorescence depends on the amount of antibody bound. Sections of chicken embryo neural retinas from 6- (A), 10- (B), 13- (C), 16- (D), and 18-day (E) embryos; (F) section from a newly hatched chick retina; (G-J) adult chicken retinas. (A-H) Sections were stained with antibody 18B8 and fluorescein isothiocyanate-conjugated anti-mouse IgG, IgA, and IgM produced in goats. (H) An oblique section through the outer synaptic layer. (1) Section was incubated with antibody synthesized by parental P3X63 Ag8 cells followed by fluorescent second antibody. The indicated layers of retina are as follows: ON, outer nuclear layer; OS, outer synaptic layer; IN, inner nuclear layer; IS, inner synaptic layer; GC, ganglion neuron layer; A, ganglion neuron axons. The bar in A represents 50 um for A-G and I and 20 gm for H.
the cell body and neurite layers of intact retina (Fig. 2G). Antigen 18B8 was most abundant in the neurite regions of aggregates (Fig. 2H). The results show that an antigen distribution similar to that observed in intact tissue may be obtained in vitro when cells are allowed to reassociate into histotypic aggregates. Further work is needed to determine how the antigen distribution is regulated. Such differences between monolayers and aggregates of retina cells have been described for expression of the enzyme glutamine synthetase (27) and for synapse differentiation (28). Characterization of Antigen 18B8. Glycolipids were extracted from chicken retina and brain and were fractionated. Antigen was found in the ganglioside fraction but not in the neutral glycolipid fraction by solid-phase radioimmunoassay. The antigen in the ganglioside fraction was destroyed by
treatment with neuraminidase (data not shown). Immunostaining of thin-layer chromatograms of gangliosides from chicken retina and brain at various stages of development revealed several antigens (Figs. 3 and 4). The major antigen in extracts of 7-, 10-, 13-, or 16-day chicken embryo retina or adult retina had the chromatographic mobility of GD1b. However, the antigen is not GD1b, as GD1b isolated from adult bovine brain did not bind the antibody. Other antigens with lower chromatographic mobilities were detected in extracts of 10-, 13-, and 16-day embryo retina.
Antibody 18B8 also detected several antigens in gangliosides from 5- to 10-day chicken embryo brain (Fig. 4B). The §Footnote Added in Proof. This antigen has been identified as ganglioside GT3 [II3 (NevAc)3LacCer] (37).
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FIG. 2. Expression of antigen 18B8 by retina cells in culture. Cells dissociated from 7 day embryonic chicken retinas were cultured in vitro for 14 days as stationary monolayers (A-F) or as cell aggregates in shaker cultures (G-H). Retina cells were cultured and stained with antibody. (A, C, and E) Phase-contrast photomicrographs; (B, D, F, and H) corresponding fields viewed by epifluorescence. (A and B) Cells were stained with control antibody synthesized by parental P3X63 Ag8 mouse myeloma cells; (C-E) cells were stained with antibody 18B8. (G) Section stained with hematoxylin and eosin viewed with bright-field optics. The bar in H corresponds to 50 jim for all panels.
pattern of antigen detected in developing brain, however, was different than that found in developing retina. An antigen with the chromatographic mobility of GD1b also was found in 5- to 10-day chicken embryo brain (Fig. 4B). Antibody 18B8 also recognized other species of gangliosides extracted from 5- to 10-day embryo brains, and these gangliosides changed during development. However, only one antigen, which exhibited a relatively low chromatographic mobility, was detected in extracts of 13- and 16-day embryo brain and adult brain. Thus, retina and brain differ with respect to both the developmental age when the low mobility forms of
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antigen 18B8 appear, and in the continued expression in the adult brain but not retina. The qualitative and quantitative changes in antigen 18B8 expression as detected by thin-layer chromatography and autoradiography occur concomitantly with the spatial and temporal changes in antigen 18B8 expression detected by immunofluorescence in tissue sections. As antigen appeared on retina cell soma during early stages of neural development, the amount of ganglioside antigen increased (embryonic days 6-10; Fig. 3). As antigen measured by immunofluorescence disappeared from cell soma and became
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Days FIG. 4. Immunostaining with antibody 18B8 of gangliosides from developing brain. (A) Lane 1, standard human brain gangliosides, -5 ,ug each; lane 2, adult chicken brain gangliosides from 18 mg (wet weight) of tissue, fractionated by thin-layer chromatography and treated with orcinol reagent to visualize the gangliosides. (B) Gangliosides extracted from 7 mg (wet weight) of 5-, 6-, 7-, 10-, 13-, or 16-day chicken embryo brain or adult brain were separated by thin-layer chromatography and antigens were detected by immunostaining. Mobilities of standard gangliosides are shown on the left.
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Proc. Natl. Acad. Sci. USA 82 4,
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FIG. 5. Immunostaining with antibody 18B8 of gangliosides from developing brain fractionated according to sialic acid content. Gangliosides were extracted from pooled 7-, 12-, and 14-day chicken embryo brains and separated into mono-, di-, tri-, tetra-, and polysialoganglioside fractions by DEAE-Sepharose column chromatography with a discontinuous gradient of potassium acetate in methanol. Gangliosides in part of the sample applied to the column and various fractions eluted from the column were fractionated further by thin-layer chromatography. Lane 1, standard gangliosides of known mobility, 5 ,ug each, visualized with orcinol reagent. In lanes 2 and 3, and each pair of lanes thereafter, are shown gangliosides visualized with orcinol reagent (even-numbered lanes) or by immunostaining (odd-numbered lanes). Gangliosides visualized with orcinol reagent were extracted from 18 mg (wet weight) of brain, whereas gangliosides visualized by immunostaining were extracted from 3.5 mg (wet weight) of brain. Lanes 2 and 3, total gangliosides before fractionation by DEAE-Sepharose column chromatography; all subsequent lanes contain ganglioside fractions eluted from the DEAE-Sepharose column with solutions containing the following concentrations of potassium acetate: lanes 4 and 5, 10 mM; lanes 6 and 7, 20 mM; lanes 8 and 9, 30 mM; lanes 10 and 11, 40 mM; lanes 12 and 13, 50 mM; lanes 14 and 15, 100 mM; lanes 16 and 17, 200 mM.
restricted to the synaptic layers, the slower migrating ganglioside antigens recognized by antibody 18B8 were expressed transiently (day 16; Fig 3). Pooled gangliosides from 7-, 12-, and 14-day chicken embryo brains were separated into mono-, di-, tri-, tetra-, and polysialoganglioside fractions by DEAE-Sepharose CL-6B column chromatography. The gangliosides in each fraction then were fractionated further by thin-layer chromatography. As shown in Fig. 5, antigens were not detected by immunostaining in the monosialoganglioside fraction (lanes 5 and 7), but were detected in the disialoganglioside fraction (lanes 9 and 11) and the tri-, tetra-, and polysialoganglioside fractions (lanes 13, 15, and 17). The fast running diffuse spot in lane 5 is due to nonspecific binding of antibody to lipid material, probably sulfatides, which often occurs when large samples are chromatographed. It is clear from Fig. 5 that the slower running antigens are polysialylated gangliosides (lanes 15 and 17). The polysialic acid content of some glycoproteins changes during neural development (29-31), and similar changes occur on gangliosides of the developing chicken brain, where gangliosides with up to seven sialic acid residues have been detected (32). The ganglioside antigens recognized by antibody 18B8 contain at least two sialyl residues but their detailed structures remain to be determined. Recently, a monoclonal antibody (anti-D1.1) was described that detects an acetylated disialoganglioside (33). In contrast to the 18B8 antigen, the D1.1 ganglioside is found on neuroblasts and disappears as neuroblasts differentiate into neurons (34). Monoclonal antibody 18B8 as well as others directed against carbohydrate determinants that discriminate among neuronal cells (11, 23, 34-36) will be useful in investigating the role of these structures in neural development. This work was supported in part by a grant from the National Institutes of Health (HL 06426) to G.B.G. 1. Chaudhari, N. & Hahn, W. E. (1983) Science 220, 924-928. 2. Kaplan, B. B. & Finch, C. E. (1982) in Molecular Approachesto Neurobiology, ed. Brown, I. R. (Academic, New York), pp. 71-98. 3. Sutcliffe, J. G., Milner, R. J., Gottesfeld, J. M. & Lerner, R. A. (1984) Nature (London) 308, 237-241. 4. Chikaraishi, D. M. (1979) Biochemistry 18, 3249-3256. 5. Hakomori, S. T. & Kannagi, R. (1983) J. Natl. Cancer Inst. 71, 231-241. 6. Panzetta, P., Maccioni, H. J. F. & Caputto, R. (1980) J. Neurochem. 35, 100-108.
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