Developmental regulation of glutamine synthetase and carbonic anhydrase II in neural retina.

Abstract

Glutamine synthetase (GS) is expressed in the neural retina only in Muller glia cells and is inducible with cortisol. A chicken genomic clone that contains at least part of the coding region for the GS enzyme was used to investigate developmental changes in the level of GS mRNA in embryonic chicken retina. A major GS transcript (-3 kilobases) detected by the probe begins to accumulate sharply on day 15 of embryonic development. When cortisol is prematurely supplied to early embryonic retina, it induces precocious accumulation of GS mRNA and of the GS enzyme. At later ages, these effects of cortisol are significantly greater, which suggests that competence to In embryonic and mature retina, GS is restricted to Muller glia cells (1). In chicken embryo retina (the most thoroughly studied system), GS level is very low until day 15-16 of development. It then begins to increase sharply in response to systemic elevation of adrenal corticosteroid hormones, and it increases in 6 days >100-fold. However, GS can be induced also precociously, already in 8-day retina, by prematurely adding a corticosteroid inducer such as cortisol to embryos or to organ cultures of isolated retina tissue (2, 3). Previous indirect evidence suggested that cortisol induced GS in the retina by eliciting GS mRNA accumulation (4, 5). Here, we used a chicken GS clone to investigate changes in the level of GS mRNA in the retina during normal development and when GS is prematurely induced.CA-II is present at high levels already in early embryonic retina (by day 5 of development) when it is found in all retina cells (6). As differentiation progresses, neurons stop expressing CA-II; by day 13, this enzyme is found only in Muller glia cells. During retina maturation, CA-II accumulates to a new high level that persists in the adult (6). It is important to note that cell proliferation in the neural retina of chicken embryo begins to decline after day 8 and ceases by day 12 of development (3). Using a CA-II cDNA clone, we investigated the accumulation and localization of CA-II mRNA during retina development in relation to changes in CA-II enzyme level. MATERIALS AND METHODSNeural Retina. Neural retina tissue was isolated under sterile conditions from eyes of chicken embryos (White Leghorn) at different stages of development and was stored at -70°C or directly used for RNA preparation.RNA Preparation and Analysis. Retina tissue was disrupted by Dounce homogenization in an ice-cold solution of 0.15 M NaCl/0.01 M Tris HCl, pH 7.5/2 mM MgCl2/0.75% Nonidet P-40. Nuclei were removed by centrifugation (8 min at 1500 x g). The supernatant was adjusted to 2.5 mM EDTA/0.5% NaDodSO4 and cytoplasmic RNA was isolated by phenol/ chloroform extraction and ethanol precipitation. Poly(A)+ RNA was selected by using an oligo(dT)-cellulose column (Collaborative Research, Waltham, MA, type III). Total RNA was prepared by the guanidinium isothiocyanate/CsCl method (7). Total RNA (10 ,tg) or poly(A)+ RNA (5 ,ug) was denatured by heating at 60°C in 2.2 M formaldehyde/50...

Glutamine synthetase protects against neuronal degeneration in injured retinal tissue

“…The embryonic chicken retina is eminently suitable for this kind of study because the level of GS in this tissue can be closely regulated by glucocorticoids (5)(6)(7)(8). Glucocorticoids regulate GS expression at the transcriptional level and induce a marked increase in GS enzyme activity in Muller glial cells (9)(10)(11)(12)(13).…”

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

“…The upstream region of the GS gene contains a glucocorticoid response element (GRE), and glucocorticoids regulate GS expression at the transcriptional level (Vardimon et al 1988;Zhang and Young 1991;Gorovits et al 1996). Supply of glucocorticoids to the embryo or to isolated retinal tissue results in accumulation of GS mRNA and protein, and in a marked increase in GS activity (Moscona 1975;Vardimon et al 1986;Patejunas and Young 1987). The hormone-mediated increase in GS expression is celltype specific: it is restricted to Mü ller glial cells, which are the only glial cells in the chicken retina and also the only cells in this tissue that express the glucocorticoid receptor protein (Linser and Moscona 1979;Gorovits et al 1994;Grossman et al 1994).…”

Molecular control of glutamine synthetase expression in the developing retina tissue

“…In contrast, at early embryonic ages, the glucocorticoid receptor is transcriptionally inactive and GS cannot be induced. Glucocorticoid receptor activity and GS inducibility increase progressively with development and by day 12, competence for GS induction is high (Moscona, 1983;Vardimon et al, 1986a;Ben-Dror et al, 1993). This was demonstrated by culturing retina tissues at different developmental ages for 24 hr in the presence or absence of cortisol and analyzing the level of GS mRNA by Northern blotting, using the [32Pl-labeled GS clone, A-GS223 (Vardimon et al, 1986a1, as a probe (Fig.…”