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The neural retina has been widely used to study the developmental patterns of ganglioside metabolism. Recent findings about in vitro differentiating chick embryo retina cells showed that: a) GD3 and GD1a ganglioside patterns undergo the most dramatic changes; b) when the cells emit neurites, GD3 ganglioside and a group of complex gangliotetraosylgangliosides (GTOG) are transiently coexpressed; c) synchronized developmental phenomena are dissociated by anti-GM1 antibodies; d) GD3 remains as a major ganglioside in differentiated neurons, though it is almost not immunoexpressed; e) GTOG affect antibody binding to GD3; f) the content of gangliosides involved in neural differentiation modifies their immunostain localization on cell membrane; g) after exogenous GTOG uptake, immature neurons mimic GD3 immunofluorescent localization of mature cells; h) a subset of purified retinal ganglion cells express GTOG characteristic of mature neurons.

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Panning-purified retinal ganglion cells (RGCs) were cultured at low density in a chemical-defined growth factor (GF)-lacking medium on substrate of different extracellular matrix (ECM) proteins. The process regrowth under these severe conditions were evaluated by morphometric measurements and by cell ELISA (CELISA) performed for neurofilaments regardless of their phosphorylated state (NF-CELISA), or for phosphorylated neurofilaments (PNF-CELISA), to respectively assess process regrowth or axonal development. The development obtained in cultures performed on laminin was taken as standard to refer the other substrata. The cellular content of Thy-1 required for panning purification as well as the gangliotetraosylganglioside (GTOG) expression and the lack of the immunolabeling of the RA4 antigen strongly suggest that the purified RGCs were mature neurons. About 80% of the 7-day-old embryo (E7)-RGCs survived 4 days in culture on any substrate, including polylysine. Conversely, E10-RGCs in about 75% of cultures on polylysine did not survive for 4 days. E7-RGCs developed better on thrombospondin and vitronectin. E10-RGCs cultured on vitronectin grew better than on laminin; on thrombospondin and collagen, E10-RGCs grew like on laminin and on fibronectin they had a poor development. The values of PNF-CELISA obtained on vitronectin, collagen and fibronectin on E7-RGC cultures were significantly higher than on laminin, which are in agreement with the longer processes observed. The flavoridin disintegrin caused a dose-response inhibition on E7-RGC cultures on thrombospondin but not on laminin, suggesting on process regrowth, the integrin-thrombospondin interaction(s) are significantly involved, while on laminin, it is the non-integrin receptor(s) which are significant involved.

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Glycogenin, the autoglucosyltransferase that primes the biosynthesis of proteoglycogen, is found in the polysaccharide linked proteoglycogen form in mammals and chicken. Glycogenin was released from proteoglycogen and its activity was measured, together with that of glycogen synthase as well as glycogen content, in muscle, liver, and brain during chicken development. The specific activity of glycogenin, expressed per protein, increased with development only in muscle and was higher than the specific activities measured in liver and brain at any time. Concomitant with the rise in activity, an enhanced expression of the protein was observed with Western blot. The specific activity of glycogen synthase increased with development in muscle and liver, while glycogen accumulation was noticeable only in liver. The results indicate that the molar concentration of proteoglycogen is higher in muscle than in liver. The high glycogen content of liver may indicate that the size of the polysaccharide moiety of proteoglycogen is larger in liver than in muscle. This is the first report of developmental modulation of de novo biosynthesis of glycogen at the level of the primer that initiates glucose polymerization.

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Both, a tailored chemically defined nutrient medium (BP5) and a sandwich culture sustain the survival for more than a week and allow the differentiation of embryonic chick retinal ganglion cells (RGCs) seeded at low density. Purification of RGCs from 7-11-day old embryos was accomplished by panning using specific anti-chicken Thy-1 antibodies immobilized in plaques. Yield of RGCs was less than 1% of the calculated number of these cells in the used retinas. This result agrees with the scarce expression of Thy-1 in immature retina; accordingly, the most mature RGCs are those probably selected by the panning. This assumption obtained support on the expression of gangliotetraoxylgangliosides (GTOG), that characterize the differentiated retinal neurons. Thus, the outgrowth of processes observed in cultured cells, might imply axonal regeneration in mature neurons. This manageable RGC culture method approaches a system for studying the in vitro trophic factors and substrata which affect axonal regrowth in central nervous system cells.

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Ganglioside expression of embryonic chick retina cells developed in vitro was analyzed by indirect immunofluorescence. Immature neurons were GD3 positive cells and the labeling was chiefly distributed all over their cell membrane. Mature neurons became GD3 negative and expressed complex gangliosides of the a- and b-pathways; nevertheless, the content of GD3 accounted for approximately 40% of the total gangliosides in these cells. Neuraminidase hydrolysis pointed out that GD3 was located in membrane of differentiated cells. The frequency of cells with the GD3 immunostain localized in restricted area of membrane of undifferentiated neurons increased significantly after adding a mixture of bovine brain gangliosides (largely complex gangliosides). Antibody binding to immobilized GD3 showed a dose-dependent inhibition by adding a mixture of bovine brain gangliosides, GM1, GD1a or asialo-GM1. Glycosphingolipids with shorter oligosaccharide chains, as cerebrosides or sulfatides, did not affect this binding. These results suggest that, concomitant with the accretion of content of complex gangliosides, a rearrangement in the membrane would occur, which progressively masks GD3 to its antibody. This rearrangement might affect putative ganglioside functions involved in neuronal differentiation.

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The immunostains of GD3, c-pathway polysialogangliosides and gangliotetraosylgangliosides belonging to the a- and b-pathways were analyzed in embryo optic lobe cells. Cells were cultured in serum free media with, and without, bovine brain ganglioside mixture (largely gangliotetraosylgangliosides). Control cells differentiated in vitro, whereas ganglioside treated cells emitted almost no neurites. In immature cells, GD3 and c-gangliosides were extensively expressed and their immunostains were observed all over the cell (general localization), whereas gangliotetraosylgangliosides were scanty and their stain was preferentially restricted to discrete areas (in clusters). In control differentiated cells, the GD3 expression was strikingly reduced and its immunostain appeared in clusters; c-gangliosides were abundant and their stain was found with general localization; the expression of gangliotetraosylgangliosides became important and their stain was observed with general localization in most of the positive cells. In ganglioside treated cells, in spite of their undifferentiated morphology, the gangliotetraosylganglioside stain appeared with general localization. These results suggest that gangliosides involved in neural ontogenesis are preferentially located in clusters when they are scarce and have general localization when they are abundant in membranes.

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Retinal cells from 7-day-old chicken embryos were cultured in the presence of a polyclonal anti-GM1 antibody, at low and high density in a "sandwich cell culture". Cells that were about 80% neurofilament positive at all times, changed their morphology and emitted processes as controls. By examining immunocytochemical expression of gangliosides, cells cultured in the presence of the antibody maintained GD3 expression longer than controls, albeit the expression of the gangliotetraosylgangliosides (GTOG) was not considerably affected. This leads to an extension of the transient period in which differentiating cells coexpressed both types of gangliosides (GD3 and GTOG). At 3-4 days in vitro the relative synthesis of GD3 was about 30% higher and that of GD1a about 40% lower than in controls, indicating a delay in the shift of the synthesis pattern. Nevertheless, the pattern of ganglioside composition resembled at 4 days in vitro. Results indicate that the anti-GM1 antibody may modulate the expression and synthesis of gangliosides without a detectable decrease in neuritogenesis. Considering that the emission of neurites occurs in coexpressing GD3 and GTOG neurons, it is suggested that neuritogenesis could be irrespective of losing the GD3 expression.

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Retina cells from 6-day-old chicken embryos were cultured in the presence of an 125I-labeled protein inhibitor of the UDP-N-acetylgalactosamine:GM3,N-acetylgalactosaminyltransferase. The cells were labeled and did not lose the incorporated radioactivity when treated with 0.125% trypsin or 1 M NaCl at 37 degrees C for 1 hr, indicating that the iodinated inhibitor was inside the cells. Immunostaining procedures using an anti-inhibitor antibody were applied to the cells cultured in the presence of the inhibitor after permeabilization of the cells. The inhibitor was found inside the round cells virtually devoid of neurites, but not in flat glial-like cells or in process-bearing neural cells. Also found was an apparent self-recovery effect of the cells for both the anti-neuritogenic effect and the modification of the pattern of labeled gangliosides produced by the inhibitor when the agent was withdrawn from the culture medium after the initial period of 20 hr. This recovery was clearly observed 72 hr after the removal of the inhibitor.

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An inhibitor of N-acetylgalactosamine:GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) from chicken blood serum, was tested for its activity on embryonic chicken neural retina in culture. The inhibitor did not change the cellular protein content of the cultures but produced a significant reduction of the labeling of gangliosides. The ratio of labeling of GD3 to GD1a increased from about 0.1 to about 0.8 in the cells cultured without or with the inhibitor, respectively. A striking effect of the inhibitor was seen on the morphology of the neurons, those cultured in its presence being practically devoid of neurites. Glial flat cells were apparently not affected.

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The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from the chick neural retina was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach, after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. In retinas from 6-day-old embryos (R6), most cells (approximately 80%) expressed GD3 but not GTC. After culturing for 7 days, (R6+7), the expression of GTC was found confined to neuron-like cells; flat cells derived from Müller cells expressed GD3 but were negative for GTC expression. On the other hand, postmitotic Müller cells obtained from 13-day-old embryo (R13) or 1-day-old hatched chick retina (RP1) expressed GD3, GM1, and polysialosyl-GTC but were unable to maintain the expression of these GTCs when kept in culture for several days. According to these results, retinal cells can be defined on the basis of their ganglioside expression as follows: (a) retinoblasts, by the expression of GD3; (b) postmitotic neuronal cells, by the expression of GTC; and (c) postmitotic Müller cells, by the expression of GD3 and GTC.(ABSTRACT TRUNCATED AT 250 WORDS)

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Cells from neural retina from 7-day chick embryos were cultured on polylysine-coated dishes up to 7 days. The small, round-shaped cells at seeding differentiated progressively, and after 4 days in vitro the majority had enlarged bodies and abundant processes. The content of protein and DNA was essentially unchanged during the entire period of culture. The incorporation of radioactivity from [3H]glucosamine into gangliosides declined slightly, reaching about 65% of the initial values at the end of the culture period. The proliferating activity measured by the incorporation of [3H]thymidine into DNA decreased to 10% or less of the initial value after 3 days in vitro. Almost at the same chronological times as in ovo, the synthesis of GD3 and of a ganglioside partially identified as GT3 decreased from 70 and 19% of the total incorporation into gangliosides in the first 20 h of culture to about 7 and 5%, respectively, after 3 days in vitro. Conversely, the synthesis of GD1a increased from about 6% at the beginning to about 70% at the end of the culture times. Immunocytochemical analyses of the expression of gangliotetraosyl gangliosides in cultured cells showed that these gangliosides appeared in the bodies and processes of cells having neuronal morphology; very little immunostaining of the scarce flattened cells, probably Müller cells, was found. The results indicate that the changes in ganglioside metabolism, which lead to decreased synthesis of gangliosides lacking the galactosyl-N-acetyl-galactosaminyl disaccharide end and to increased synthesis of gangliotetraosyl gangliosides, occur in cells that in culture differentiate into neurons.

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Some properties of the uridine-5'-diphospho-N-acetylgalactosamine:hematoside N-acetylgalactosaminyltransferase were studied in retina tissue from chick embryos at 7 and 14 days of development. The Vmax was about 6-fold higher in retinas from 14 day embryos than in retinas from 7 day embryos. No differences were found either in the apparent Michaelis constant for both donor nucleotide and acceptor glycolipid, or in the optimal detergent concentration, or in the stability upon storage at -14°C or heating at 50°C. Mixtures of homogenates of retinas from 7 day and from 14 day embryos gave the activity values expected for samples free of effectors diffusible and in excess. From experiments of partial delipidation of retina homogenates and reconstitution with lipid from retina homogenates from one or the other age, no indications were found that the activity was modulated by developmental changes in the lipid environment of the enzyme. Taken together, the results suggest that the increase of activity during development was not due to qualitative changes in the catalytic characteristics of the enzyme.