Hormonal induction of glutamine synthetase in cultures of embryonic retina cells: requirement for neuron-glia contact interactions

Cortisol induces glutamine synthetase (GS) in gliocytes of chick embryo neural retina. Using adherent cultures of retina cells we have demonstrated that responsiveness of the gliocytes to GS induction by the hormone requires contact with neurons. GS is not inducible in high-density cultures depleted of neurons and consisting only of gliocytes. In neuron-containing cultures, induced GS was detected immunohistochemically only in those gliocytes that were closely juxtaposed with clusters of neurons. Unlike the induction of GS, the expression of carbonic anhydrase-C (which does not require cortisol) persisted in these glia cells also in the absence of neurons. The nature and role of glia-neuron interactions in the hormonal induction of GS are briefly discussed.     

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

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.     

Effects of cytosine arabinoside on differential gene expression in embryonic neural retina. II. Immunochemical studies on the accumulation of glutamine synthetase

Cytosine arabinoside (Ara-C) elicits a significant increase in the level of the enzyme glutamine synthetase (GS) while it markedly reduces overall RNA and protein synthesis in cultures of embryonic chick neural retina. This increase was analyzed by radioimmunochemical procedures and compared with the induction of GS by hydrocortisone (HC). Accumulation of GS in Ara-C-treated retinas was found to be due to de novo synthesis of the enzyme; however, unlike the induction of GS by HC, Ara-C caused no measurable increase in the rate of GS synthesis. The results indicate that Ara-C facilitates GS accumulation largely by preventing degradation of the enzyme. Even though Ara-C inhibits the bulk of RNA synthesis in the retina, it does not stop the formation of GS-specific RNA templates. However, the progressive accumulation of these templates does not result in an increased rate of GS synthesis unless Ara-C is withdrawn from such cultures under suitable experimental conditions. Thus, it is suggested that the continuous presence of Ara-C imposes a reversible hindrance at the translational level which limits the rate of GS synthesis. The results demonstrate that the increase in retinal GS elicited by Ara-C is achieved through mechanisms which are quite different from those involved in the hydrocortisone-mediated induction of this enzyme.     

Glutamine synthetase induction in embryonic neural retina. Interactions of receptor-hydrocortisone complexes with cell nuclei.

In the neural retina of the chick embryo, hydrocortisone (HC) elicits differential gene expression resulting in the induction of glutamine synthetase (GS), which is an enzyme marker of differentiation in the retina. The relationship between nuclear binding of receptor-hydrocortisone (R-HC) complexes and GS induction was investigated in cultures of retina tissue from 12-day chick embryos. The number of HC binding sites in the cytoplasm was estimated as 1650+/-200 per retina cell; there are approximately 1500+/-100 acceptor sites for R-HC per retina nucleus. GS induction in the retina became detectable only after R-HC bound to more than 40% of the nuclear acceptors sites; increased binding coincided with higher induction levels, until complete site saturation was attained; Proflavine, which blocks preferentially and completely GS induction in the retina by interfering in the nucleus with the enzyme-inducing action of the hormone, reduced nuclear binding of R-HC by only 20%; thus, only part of the R-HC that binds in the nucleus appears to be directly involved in eliciting the induction of GS. Within one hour after exposure of the retina to an inducing dose of HC, there was translocation of HC and HC-receptors (as R-HC complexes) from the cytoplasm into the nucleus and saturation of nuclear accepegan to decline; in 12 h, it was reduced to 50% of the initial saturation level. Since, during this time, the enzyme activity to increase, persistence of the induced state depends on association of the hormone with only a portion of the sites in the nucleus to which it can bind. The decrease in the amount of bound HC in the nuclei of induced cells was accompanied by an increase in the level of HC receptors in the cytoplasm. About 50% of this increase could be prevented by cycloheximide; this suggests that the reappearance of HC receptors in the cell cytoplasm may be due, at least in part, to de novo synthesis of HC receptors.     

Does cyclic-3′,5′-AMP induce glutamine synthetase in embryonic neural retina?

The inducibility of retinal glutamine synthetase (GS) by dibutyryl cyclic-3′,5′-AMP (DB-cAMP) was re-examined in view of conflicting reports. Various lots of DB-cAMP were compared for a) ability to induce GS in cultures of embryonic chick neural retina, and b) their composition as visualized by paper chromatography. Chromatographically purified DB-cAMP did not induce retinal GS, nor did cAMP, DB-cGMP, epinephrine, or norepinephrine; none of these enhanced the induction of GS by hydrocortisone. Some of the agents occasionally caused small increases in GS activity; however, these were invariably below the GS levels induced consistently by hydrocortisone. A single lot of DB-cAMP was found which significantly raised GS activity in the retina; it contained a contaminant which when isolated was found to be responsible for this effect.     

The Development of Inducibility for Glutamine Synthetase in Embryonic Neural Retina: Inhibition by BrdU

The hydrocortisone-mediated induction of glutamine synthetase (GS) in the neural retina of the chick embryo is a characteristic and unique feature of differentiation of this tissue. The induction involves genomic activity elicited by the inducer resulting in synthesis and accumulation of the enzyme. We describe correlations between the growth of embryonic retina tissue in vivo and in vitro and the development of its inducibility for GS, and demonstrate that this development proceeds through two phases: competence-acquisition phase (before the 7th day of development), and maturation phase. BrdU applied for 24 h to retinas of 5-day embryos irreversibly suppresses the development of induction-competence. However, BrdU does not affect the progressive maturation of inducibility when applied to retinas that already are fully induction-competent (8 days and older). The short treatment with BrdU of 5-day retinas also causes defective histogenesis resulting in drastic malformation of the tissue. The nature of the processes involved in competence-acquisition and in the maturation of inducibility for GS are examined. Possible mechanisms by which BrdU prevents the development of induction-competence for GS in the early embryonic retina and elicits defective histogenesis are discussed.     

Glutamine synthetase enhances the clearance of extracellular glutamate by the neural retina

Clearance of synaptic glutamate by glial cells is required for the normal function of excitatory synapses and for prevention of neurotoxicity. Although the regulatory role of glial glutamate transporters in glutamate clearance is well established, little is known about the influence of glial glutamate metabolism on this process. This study examines whether glutamine synthetase (GS), a glial-specific enzyme that amidates glutamate to glutamine, affects the uptake of glutamate. Retinal explants were incubated in the presence of [(14)C]glutamate and glutamate uptake was assessed by measurement of the amount of radioactively labeled molecules within the cells and the amount of [(14)C]glutamine released to the medium. An increase in GS expression in Müller glial cells, caused by induction of the endogenous gene, did not affect the amount of glutamate accumulated within the cells, but led to a dramatic increase in the amount of glutamine released. This increase, which was directly correlated with the level of GS expression, was dependent on the presence of external sodium ions, and could be completely abolished by methionine sulfoximine, a specific inhibitor of GS activity. Our results demonstrate that GS activity significantly influences the uptake of glutamate by the neural retina and suggest that this enzyme may represent an important target for neuroprotective strategies.     

Histogenesis and dependent glutamine synthetase inducibility in embryonic neural retina. Irreversible inhibition of differentiation by 5-bromodeoxyuridine

Young, mitotically active neural retinas from 7-day chick embryos were cultured with 5-bromodeoxyuridine (BrdU) for 8 hr or more. After this treatment, they failed to differentiate beyond the stage at which they were explanted; there was no histogenesis or increase in glutamine synthetase (GS) inducibility in intact tissues or in aggregates of dissociated cells. Normally GS can be induced in the retina with hydrocortisone as the cells cease to be mitotically active and begin showing histological organization after day 7. This inhibition by BrdU was irreversible even in the presence of excess thymidine. Overall incorporation of ¹⁴C-amino acids into protein was only slightly inhibited, and the ability of cells from treated tissue to aggregate and sort out from nonneural cell types was unaffected. Control cultures without BrdU showed considerable histogenesis and a parallel increase in enzyme inducibility. Postmitotic 10-day retinas appeared to be unaffected by BrdU. The incorporation rates of both tritiated BrdU and thymidine (dT) into DNA were 14× higher in 7- than in 10-day retinas. Simultaneous addition of excess unlabeled dT with either of the labeled nucleosides reduced their incorporation and reduced the inhibitory action of BrdU on differentiation.It is concluded that BrdU irreversibly inhibits the differentiation of retina cell surface properties involved in histogenesis and dependent cytodifferentiation without affecting already differentiated properties of the cell surface. The results support the hypothesis that histogenesis is directed by genes affecting specific cell surface properties.     

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

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.     

Cortisol receptors and inducibility of glutamine synthetase in embryonic retina

Glutamine synthetase (GS) is a marker enzyme for Müller glia cells in neural retina. In chick embryo retina GS begins to increase sharply on the 16th day of development, but can be precociously induced by premature supply of the inducer, cortisol, already on the 8th day. At this stage GS inducibility is low, but it increases progressively with embryonic age. We investigated whether there was a corresponding age-dependent increase of cortisol-binding molecules (cortisol receptors) and found that their level is highest in the early retina and decreases with development. In light of this inverse relationship, we examined whether functional characteristics of these receptors change with age, but detected no differences. In in vitro tests, receptors from older retina translocated cortisol into nuclei from young retina, and vice versa, with similar effectiveness. Also, cortisol receptors from liver cells (which differ from retina receptors) can translocate the hormone into retina nuclei, and vice versa. These findings indicate that translocation of cortisol receptors is neither tissue-specific or age-dependent, nor is it conditional on the total amount of receptors normally present in cells. Therefore, the age-dependent increase of GS inducibility in embryonic retina cannot be directly related to quantitative or functional differences of cortisol receptors and is evidently controlled primarily at the gene level. The very large amount of cortisol-binding molecules in early embryonic retina raises the possibility that they play some role in early differentiation of retina cells unrelated to hormone binding.     

Nuclear Binding of Hydrocortisone-Receptors in the Embryonic Chick Retina and its Relationship to Glutamine Synthetase Induction

Binding of hydrocortisone (HC) to cytoplasmic receptors andof the resulting receptor-hydrocortisone complex (R-HC) to nuclearacceptor sites has been studied in neural retina cells of thechick embryo, in which this hormone induces glutamine synthetase(GS). These studies were done in a cell-free system, as wellas in intact retina tissue in culture. Optimal conditions, specificity, and the quantitative aspectsof R-HC binding to nuclei in the cell-free system were determined.Isolated nuclei retained their binding specificity for R-HCprepared from retina cytosol; at saturation, the total numberof nuclear acceptor sites for R-HC was estimated to be in therange of 1500 per nucleus. These sites were resistant to RNAsebut sensitive to DNAse. When retina tissue was cultured in the presence of progressivelyhigher doses (0–90 nM) of HC, increasing amounts of receptorswere translocated from the cytoplasm to the nuclei. Followingincubation of retina with 9–90 nM HC, the cytoplasm wasalmost completely depleted of receptors and the nuclei becamesaturated with R-HC. From the amount of R-HC bound to nucleiat saturation, the estimated total number of R-HC acceptor sitesper nucleus was comparable to that derived from the cell-freesystem. Increases in the level of GS induction in the retinacorrelated well with amounts of R-HC complex bound by nuclei.     

Glycerol Phosphate Dehydrogenase in Developing Chick Retina and Brain

Abstract: The development of cytoplasmic glycerol phosphate dehydrogenase (GPDH) activity in chick neural retina is compared with that in brain. GPDH converts dihydroxyacetone phosphate to glycerol 3-phosphate, an intermediate in phospholipid synthesis. The enzyme is known to be under corticosteroid control in rat brain and spinal cord (but not muscle or liver) and in primary oligodendrocyte cultures. It has not been previously studied in the eye. In chick brain the GDPH specific activity rises fivefold from the early embryo to the adult, with nearly all the increase occurring between embryonic day 14 and hatching. This time course correlates well with the known maturation of chick adrenal cortex (which produces corticosteroids). On the other hand, in chick retina the GPDH specific activity remains at a low basal level throughout development. Furthermore, adult rat and beef retinas show much lower enzyme activity than do the corresponding brain tissues. GPDH can be induced precociously by hydrocortisone in embryonic chick brain from days 12 through 16, both in the intact embryo and in tissue culture; however, GPDH is not at all inducible in chick retina. The developmental increase in chick brain GPDH can be correlated qualitatively with myelin formation, as shown by luxol fast blue staining, whereas no myelin is seen in retina at any age. Our results are consistent with recent immunocytochemical studies demonstrating that GPDH in rat brain is associated with myelin-producing oligodendroglial cells, absent in retina. In comparison, another glial enzyme, glutamine synthetase (GS), known to be inducible in both chick brain and retina, is localized in brain astrocytes and retinal Müller cells.     

Establishment of the scleral cartilage in the chick.

This study was undertaken to investigate the establishment of the scleral cartilage in the chick embryo. Johnston et al. (1974) has demonstrated that most of the cells of the scleral cartilage originate in the cranial neural crest. By means of a series of chorioallantoic grafts of pigmented retina, and its adherent periocular mesenchyme from stage 11 to 25, the present experiments show that the cranial neural crest cells arrive at the eye in sufficient numbers to form cartilage by stage 14. Pigmented retina, denuded of mesenchyme, from stage 16 embryos implanted into the head of stage 13 embryos induces cartilage formation in head mesenchyme. However, neither pigmented retina nor spinal cord could induce cartilage formation in chorioallantoic mesenchyme. Combination grafts of cranial neural crest and presumptive optic vesicle developed neural tissue, pigmented retina, and in some cases sclera-like cartilage. Thus, periorbital mesenchyme, derived largely from cranial neural crest, at about stage 14 develops the scleral cartilage in response to induction by the pigmented retina.     

A re-examination of lens induction in chicken embryos: in vitro studies of early tissue interactions

Early studies on lens induction suggested that the optic vesicle, the precursor of the retina, was the primary inducer of the lens; however, more recent experiments with amphibians establish an important role for earlier inductive interactions between anterior neural plate and adjacent presumptive lens ectoderm in lens formation. We report here experiments assessing key inductive interactions in chicken embryos to see if features of amphibian systems are conserved in birds. We first examined the issue of specification of head ectoderm for a lens fate. A large region of head ectoderm, in addition to the presumptive lens ectoderm, is specified for a lens fate before the time of neural tube closure, well before the optic vesicle first contacts the presumptive lens ectoderm. This positive lens response was observed in cultures grown in a wide range of culture media. We also tested whether the optic vesicle can induce lenses in recombinant cultures with ectoderm and find that, at least with the ectodermal tissues we examined, it generally cannot induce a lens response. Finally, we addressed how lens potential is suppressed in non-lens head ectoderm and show an inhibitory role for head mesenchyme. This mesenchyme is infiltrated by neural crest cells in most regions of the head. Taken together, these results suggest that, as in amphibians, the optic vesicle cannot be solely responsible for lens induction in chicken embryos; other tissue interactions must send early signals required for lens specification, while inhibitory interactions from mesenchyme suppress lens-forming ability outside of the lens area.     

Functional Identification of Cell Phenotypes Differentiating from Mice Retinal Neurospheres Using Single Cell Calcium Imaging

Degeneration of neural retina causes vision impairment and can lead to blindness. Neural stem and progenitor cells might be used as a tool directed to regenerative medicine of the retina. Here, we describe a novel platform for cell phenotype-specific drug discovery and screening of proneurogenic factors, able to boost differentiation of neural retinal progenitor cells. By using single cell calcium imaging (SCCI) and a rational-based stimulation protocol, a diversity of cells emerging from differentiated retinal neurosphere cultures were identified. Exposure of retinal progenitor cultures to KCl or to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) stimulated Ca²⁺ transients in microtubule-associated protein 2 (MAP-2) positive neurons. Doublecortin (DCX) and polysialated neural cell adhesion molecule (PSA-NCAM) positive neuroblasts were distinguished from differentiated neurons on the basis of their response to muscimol. Ca²⁺ fluxes in glial fibrillary acidic protein (GFAP) or glutamine synthetase (GS) positive cells were induced by ATP. To validate the platform, neurospheres were treated with brain-derived neurotrophic factor (BDNF) (proneurogenic) or ciliary neurotrophic factor (CNTF) (gliogenic factor). BDNF increased the percentage of differentiated cells expressing Tuj-1 sensitive to KCl or AMPA and reduced the population of cells responding to muscimol. CNTF exposure resulted in a higher number of cells expressing GFAP responding to ATP. All together, our data may open new perspectives for cell type-specific discovery of drug targets and screening of novel proneurogenic factors to boost differentiation of neural retina cells to treat degenerative retinal diseases.     

Specific alteration of NCAM-mediated cell adhesion by an endoneuraminidase

A phage endoneuraminidase that specifically cleaves alpha-2, 8-linked polysialic acid has been found to be a useful probe for examining the biological role of this sugar moiety on the neural cell adhesion molecule (NCAM). The enzyme caused a 3.3-fold increase in the rate of NCAM-dependent aggregation of membrane vesicles from chicken embryonic brain, without the nonspecific effects previously encountered with the use of exoneuraminidases. The enhancement of aggregation was closely correlated with removal of sialic acid as assessed by electrophoretic mobility. Extension of this analysis to cultures of spinal ganglia indicated that removal of sialic acid by the endoneuraminidase results in an increase in the thickness of neurite bundles. This enhancement of fasciculation was reversed by addition of anti-NCAM Fab, suggesting that the enzyme treatment was not toxic and did not produce nonspecific effects on adhesion. Injection of the enzyme into the eyes of 3.5-d chicken embryos consistently produced a striking array of abnormalities in those parts of the neural retina that contained the highest concentrations of NCAM at the time of injection. These perturbations included a dramatic thickening of the neural epithelium in the posterior eye, a failure of cells in this region to elongate radially, formation of an ectopic optic fiber layer, and an incomplete association of the presumptive pigmented epithelium with the neural retina. These results provide the first direct evidence that the polysialic acid on NCAM has a regulatory effect on adhesion between living cells, and that the amount of this carbohydrate is critical for the normal morphogenesis of nerve tissue.