A.A. Moscona

Cellular compartmentalization of carbonic anhydrase-C and glutamine synthetase in developing and mature mouse neural retina

Using immunohistochemical methods, we have determined the cellular localization of the enzymes, glutamine synthetase (GS) and carbonic anhydrase-C (CA-C), in mouse neural retina during development and in the mature tissue. GS is always confined exclusively to the Müller glial cells; it is first detectable in these cells post-natally on about day 12, i.e. shortly before the eyes open. Also CA-C in the mature retina is localized in the Müller cells but, in addition, it is found in certain amacrine neurons as well. CA-C is first detectable in the retina already several days before birth; at that time it is found in most of the cells, with the exception of the emerging ganglion cells. However, with advancing differentiation, CA-C becomes progressively restricted to Müller cells and to a sub-category of amacrine neurons, and persists only in these cells in the mature retina. The present results extend our previous studies on these enzymes in the avian retina; they demonstrate that also in mammalian retina, different temporal and cellular patterns of GS and CA-C expression and localization earmark distinct phases of structural and functional differentiation of the retina. The striking developmental changes in the cellular localization of CA-C, and the finding of this enzyme in certain amacrine neurons as well as in Müller cells, raise questions about the role of CA-C in the retina, and about mechanisms regulating its expression in specific cell types.

Induction of glutamine synthetase in embryonic neural retina: its suppression by the gliatoxic agent α-aminoadipic acid

Competence for cortisol-mediated induction of glutamine synthetase (GS) is a differentiation marker of embryonic neural retina. Earlier work has indicated that the induction and accumulation of GS is localized in the Müller glia cells. This localization was presently confirmed by the finding that the gliatoxin D,L-alpha-amino-adipic acid (AAA) reduces responsiveness to GS induction by 60--90% due to preferential damage to Müller cells. The tests were performed on organ cultures of retina tissue from chick embryos, and on retina cell aggregates in which there is tissue reconstruction. The presence of GS-inducible Müller cells was monitored by immuno-staining of tissue sections with anti-GS antiserum. Reduction of GS inducibility due to pretreatment with AAA resulted in virtual absence of cells that immunostained for GS. The preferential toxicity of AAA for Müller cells was also demonstrated by cell viability tests; it was further corroborated by the finding that treatment with AAA greatly reduced the level of carbonic anhydrase activity, another enzyme localized predominantly in Müller cells, but did not affect gamma-aminobutyric acid transaminase and choline acetyl transferase, neuronal enzymes. Susceptibility of Müller cells to AAA was found to increase with embryonic development of the retina. We suggest that acquisition of susceptibility for AAA represents another differentiation marker of embryonic Müller cells.

Lentoids in aggregates of embryonic neural retina cells

Abstract Cells dissociated from neural retinas of 13 and 16-day chick embryos were precultured in monolayer for 7 days and then aggregated by rotation. After 2 days, the aggregates contained lentoidal structure which immunostained with anti-lens antiserum. Our results suggest that the lentoidogenic cells were derived from retinal glia cells modified by disruption of their normal associations with neurons, and/or by precultivation in monolayer. Cell aggregation expedited formation of contacts between these cells and expression of their modified phenotype. The approach described here lends itself to detailed studies on various aspects of lentoidogenesis from neural retina cells.

Induction of glutamine synthetase in embryonic neural retina in vitro by inhibitors of macromolecular synthesis

Abstract In the embryonic neural retina glutamine synthetase (GS) is induced by hydrocortisone and related 11β-hydroxycorticosteroids which cause the accumulation of stable, active templates for the synthesis of this enzyme. It has now been shown that, under suitable experimental conditions GS can be induced by exposure of the isolated retina to cytosine arabinoside, or by treatment with cycloheximide followed by inhibition of transcription. It is suggested that these agents induce GS in the retina by blocking the production of inhibitor(s) which in the uninduced cells prevent the accumulation of stable, active templates for this enzyme.

Stimulation of DNA synthesis by ouabain and concanavalin A in cultures of embryonic neural retina cells.

Ouabain and concanavalin A, agents which bind to specific sites in the cell membrane, stimulate DNA synthesis and cell replication in monolayer cultures of neural retina cells from late chick embryos. The results suggest a relationship between control of retina cell replication and properties of the cell membrane. The experiments involved measurements of 3H-thymidine incorporation in primary monolayer cultures (24-48h) of retina cells from embryos of different ages. Stimulation by ouabain was greatest in cells from 14-day embryos, and its magnitude was similar to that elicited in these cell cultures by concanavalin A. Simultaneous treatment of 14-day retina cells with both agents resulted in a greater than additive stimulation of DNA synthesis. Our results demonstrated that, although during normal embryogenesis cell replication in the neural retina has virtually ceased by day 14 of development, some cells retained a capacity for mitogenesis when exposed to conditions such as provided in these experiments. By autoradiography the responding cells were identified as large epithelioid retina cells (LER cells). Under optimal conditions of simultaneous treatment with ouabain and Con A about 20% of the LER cells showed stimulation of DNA synthesis. The nature of LER cells and other aspects of our findings are discussed.

Involvement of poly-A in selective gene expression: Suppression of enzyme induction in neural retina by inhibitors of poly-A synthesis

Abstract The hormonal induction og glutamine synthetase (GS) in embryonic neural retina involves selective accumulation of stable and active RNA templates for GS synthesis. Cordycepin, (3′-deoxyadenosine) suppresses this induction in close correlation with its inhibition of poly-adenylate (poly-A) synthesis. Similarly ethidium bromide, which also reduces poly-A synthesis, suppresses the induction of GS. Both agents inhibit GS induction by acting at a pre-translational level. The overall results suggest that formation of poly-A is required for the induction of GS; they provide the first indication of a relationship between poly-A synthesis and a specific, inducible, gene-controlled aspect of cell differentiation.

Comparative characterization of monoclonal antibodies to carbonic anhydrase.

Monoclonal antibodies (Mabs) were generated to avian carbonic anhydrase-C and characterized; their reactivity with human, murine, bovine, chicken and fish erythrocyte carbonic anhydrase-C, and with human carbonic anhydrase-B was investigated by ELISA and electroblot techniques. Reactivity of the Mabs with native and SDS-denatured carbonic anhydrase was compared. Mabs that recognize antigenic determinants shared by all the carbonic anhydrases examined were identified. The results demonstrate the potential usefulness of these particular probes for investigating various aspects of function, evolution, development and regulation of this important, but not well understood group of enzymes.

Modulation of cortisol receptors in embryonic retina cells by changes in cell-cell contacts: Correlations with induction of glutamine synthetase

Abstract Cortisol induces glutamine synthetase (GS) in neural retina tissue of chick embryos. GS induction represents a characteristic feature of embryonic retina differentiation. However, if the tissue is dissociated into single cells, the dispersed cells are not inducible for GS. We report that cell dispersion results in a rapid and marked reduction in the level of cortisol-binding cytoplasmic receptors. This reduction persists if the cells are maintained in a dispersed state. However, if the cells are reaggregated and they reconstruct tissue-like contacts and architecture, the level of cortisol receptors increases, and so does inducibility for GS. The results indicate that, in the embryonic neural retina histotypic cell contacts and interactions are involved in regulating the level of cortisol receptors. We propose that cell contact-dependent signals from the cell surface may modulate levels of cytoplasmic cortisol receptors necessary for GS induction.

Development of differential affinities and positional information in embryonic retina cells: Inhibition by BrdU

Embryonic neural retina cells fail to develop surface properties for type-specific cell recognition if they are transiently exposed to BrdU during an early, critical age. Such cells do not proceed with histogenetic positioning, organization and differentiation and, instead, form a malformed cell mass. This effect of BrdU is correlated with BrdU incorporation into DNA, and it can be prevented by simultaneous treatment of the cells with cytosine arabinoside. The proposed working hypothesis is that, in this system, BrdU interferes with expression of genes controlling cognitive specification of the cell surface.

Binding of receptor-hydrocortisone complexes to isolated nuclei from embryonic neural retina cells.

Hydrocortisone (HC) induces glutamine synthetase in the embryonic chick neural retina. The binding of cytoplasmic receptor-hydrocortisone (R-HC) complexes to isolated retina nuclei has been studied in a cell-free system. Optimal conditions, specificity and quantitative aspects of binding were determined. The isolated nuclei retained binding specificity for the R-HC complex prepared from retina cytosol. Free HC, estradiol-receptor complexes from retina cytosol and HC-receptor complexes from mouse brain cytosol or from chick serum did not bind to the nuclei. Assuming monovalency of the binding sites, the number of nuclear acceptor sites per retina cell for the R-HC complex was estimated to be in the range of 1500. These sites were resistant to RNAse but sensitive to DNAse.