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We have compared the steroid responsiveness of Müller glial cells of intact embryonic chicken retina with that of primary cultures derived from Müller glia. Appropriately constructed fusion genes were found to be highly glucocorticoid inducible after their cotransfection with an expression vector encoding the human glucocorticoid receptor (GR) into intact embryonic d-10 (E10) or E5.5 retina. Dramatically attenuated inductions were obtained after contransfection of Müller cell primary cultures. The progesterone receptor (PR) was also demonstrated to function in intact retina, but not in Müller cell primary cultures. An immunochemical assay was utilized to confirm that a glucocorticoid-responsive, beta-galactosidase-encoding fusion gene was specifically induced in Müller cells after its transfection into intact retina. Thus, in contrast to Müller cells in intact retina, Müller cells in primary culture have lost the capacity to achieve transcriptional activation by steroid receptors. We postulate that coordinate expression of the GR, and other more general factors required for steroid inducibility, is lost by dispersion and primary culture of retinal Müller glial cells.

This article demonstrates that the chicken glutamine synthetase (GS) promoter contains cis-acting elements that direct transcription to retinal Müller glial cells. The transient assay system developed to identify these elements involved electroporation of intact retinal tissue with GS-beta-galactosidase fusion genes followed by preparation of primary cultures and histochemical assay of cells expressing beta-galactosidase. Plasmids containing beta-galactosidase under transcriptional control by two different viral promoters are expressed primarily in neuronal cells after transfection of intact embryonic d 12 retina. In sharp contrast, expression is primarily in Müller glia after transfection with a GS-beta-galactosidase fusion gene. Although GS is glucocorticoid inducible, steroid hormone is not required to achieve Müller cell-selective expression of the GS-beta-galactosidase fusion gene. Deletion studies indicate that multiple cis-acting elements located between nucleotides-436 and -61 relative to the GS transcription start site contribute to produce Müller cell selectivity. Moreover, these upstream elements enhance expression of a heterologous promoter in Müller cells not neurons. These results indicate that an enhancer located between 61 and 436 nucleotides upstream of the transcription start site contributes to Müller cell-selective expression of the GS gene in the retina.

This report establishes that increasing the activity of cyclic AMP-dependent protein kinase (protein kinase A; PKA) potentiates glucocorticoid-mediated signaling in embryonic day 5.5 (E5.5) chicken retina. Expression of a glutamine synthetase-chloramphenicol acetyltransferase (CAT) fusion gene is not induced by treatment with glucocorticoid hormone in transfected E5.5 retina. However, treatment of the retina with forskolin, an activator of adenyl cyclase, or cotransfection with an expression vector encoding PKA is sufficient to render the fusion gene hormonally responsive. Similar results are obtained after forskolin treatment of E5.5 retina that have been transfected with a plasmid that contains the CAT reporter gene under transcriptional control by the thymidine kinase promoter and a 46-nucleotide enhancer with two glucocorticoid response elements (GREs). In contrast, forskolin augments but is not required to achieve glucocorticoid-inducible CAT gene expression in E5.5 retina transfected with a plasmid that contains the reporter driven by a minimal promoter with six juxtaposed GREs. Based on these results, we postulate that E5.5 retina contain glucocorticoid receptors whose signal transduction properties are enhanced by PKA. Unlike the transiently expressed glutamine synthetase fusion gene, however, activation of PKA does not render the endogenous glutamine synthetase gene glucocorticoid-inducible. Thus, its expression appears to be subject to an additional level of control in the developing retina.

Although glucocorticoid receptors are present throughout retinal development, the chicken glutamine synthetase (GS) gene becomes hormonally inducible between embryonic day 7.5 (E7.5) and E8. In this report we demonstrate that a transiently expressed GS-chloramphenicol (CAT) fusion gene is subject to temporal control resembling that experienced by the endogenous GS gene during in vitro retinal development. In addition, an enhancer located approximately 2 kilobases upstream of the GS transcription start site renders an SV40-CAT fusion plasmid hormonally inducible in E10, but not E5.5 retina, thereby implicating this element as the mediator of developmentally regulated expression. The enhancer contains a single glucocorticoid-response element juxtaposed to an essential ancillary site. Band shift assays demonstrate that nuclear extracts obtained from E5.5, E10, and E18 retina all contain similar levels of proteins that interact with the ancillary site, suggesting that developmental regulation of the hormonal response does not reflect the timed appearance of an ancillary factor. However, supplying exogenous glucocorticoid receptor through cotransfection is sufficient to produce hormonally inducible expression of the glutamine synthetase-CAT fusion gene in nonresponsive early stage retina. Based on these data, we postulate that the general glucocorticoid signaling pathway is compromised in early stage retina in a manner that is compensated by overexpression of the glucocorticoid receptor.

The cis-acting element mediating glucocorticoid inducibility of the chicken glutamine synthetase gene has been identified. Transfection studies using intact embryonic chicken retinae and L6 myoblasts demonstrate that sequences located between 1,849 and 2,120 nucleotides upstream of the glutamine synthetase transcription start site are required to achieve hormonally inducible expression of the chloramphenicol acetyltransferase gene. Moreover, a 42-nucleotide fragment from this region provides a robust hormonal induction when positioned approximately 2 kilobases from the SV40 early promoter. A sequence containing 8 nucleotides in common with the 12-nucleotide consensus glucocorticoid response element is encoded within this element. DNase I footprinting experiments confirm that this consensus sequence provides the only binding sites for the glucocorticoid receptor within the DNA required for inducibility. Removal of 8 nucleotides that map outside of the footprinting region results in attenuation of the hormonal response in L6 myoblasts and abolition of the response in embryonic retinae. This deletion eliminates the sequence 5'CAGCGTCA3', a sequence that resembles the canonical cyclic AMP response element (CRE), activating transcription factor (ATF), and AP1 binding sites. These results suggest that the glucocorticoid receptor acts in collaboration with a member of the jun/fos/ATF/CREB family of transcription factors to mediate a strong glucocorticoid induction at a site 2.1 kilobases upstream of the glutamine synthetase transcription start site.

This report correlates constitutive and glucocorticoid-mediated alterations in glutamine synthetase gene expression observed in chicken retinal organ cultures with activation of the gene during terminal differentiation. Because glucocorticoid hormones are potent inducers of glutamine synthetase in embryonic retinae, they have been considered as primary mediators of the developmental rise. However, unlike what might be expected of a developmental inducer, our data demonstrate that glucocorticoids act as reversible regulators of glutamine synthetase transcription. Moreover, long-term organ culture of embryonic retina in the absence of glucocorticoids leads to a dramatic increase in expression of glutamine synthetase mRNA. These studies argue that although glucocorticoids may potentiate transcription of glutamine synthetase, the retina has been programmed as early as embryonic day 6 to express this enzyme at a later time via a glucocorticoid-independent process. Interestingly, transcription of retinal glutamine synthetase increases by only approximately 10-fold during development, based on nuclear run-on measurements. Therefore, post-transcriptional control mechanisms might also contribute to the 130-fold increase in glutamine synthetase mRNA observed during terminal differentiation.

In this communication we demonstrate that gene transfer methodology can be applied to study gene expression in intact retinal explant cultures. The appropriate enzyme activity is observed in extracts obtained after electroporation of embryonic day-10 chicken retina with plasmids containing the chloramphenicol acetyltransferase-encoding or beta-galactosidase-encoding reporter genes under transcriptional control by the Rous sarcoma virus long terminal repeat. Similar results are obtained using Ca.phosphate-mediated gene transfer. Moreover, it has been previously established that glucocorticoid hormones stimulate transcription of glutamine synthetase (Glns) mRNA in embryonic retina. We report here that, based on the results of gene transfer experiments with chimeric plasmids containing 5'-flanking DNA derived from the cloned chicken Glns-encoding gene (Glns), essential glucocorticoid response elements reside between approx. 1.3 kb and 2.5 kb upstream from the Glns transcription start point. These data show that transfection of explant cultures can provide a useful approach to the study of gene expression in complex systems.

We have characterized the glucocorticoid hormone induction of glutamine synthetase mRNA in embryonic chick retinal organ cultures by quantitative dot hybridization using a cDNA clone derived from chick retinal RNA. Hydrocortisone (Kapp = 3-4 nM) and dexamethasone (Kapp = 1-2 nM) produce an approximate 30-fold increase in glutamine synthetase mRNA after incubation of organ cultures derived from embryonic day 12 retinae with either hormone for 3 hr. Progesterone is a poor inducer. The glucocorticoid-mediated rise is rapid (t1/2 = 2-3 hr) and occurs in the presence of either of the protein synthesis inhibitors cycloheximide or puromycin, indicating that the induction is a primary or direct response to the hormone. However, the magnitude of the hormonal response observed in culture increases markedly during retinal development. These observations, coupled with the previously reported absence of a hormonal induction in embryonic liver, raise the possibility of a synergistic mechanism, involving tissue-specific regulatory molecules in addition to the glucocorticoid hormone receptor, to explain the retinal-specific primary glucocorticoid hormone induction of glutamine synthetase mRNA.

Complementary DNA (cDNA) and genomic clones encoding chicken glutamine synthetase (Glns) have been isolated. The nucleotide (nt) sequence of the 2728-bp cDNA specifies a 91-nt 5' untranslated sequence, a 1119-nt open reading frame, and a 1518-nt 3' untranslated sequence that contains several A + T-rich regions but lacks a canonical endonucleolytic-cleavage/polyadenylation signal. Based on sequence analysis of the cloned gene, the Glns transcription unit spans 7.0 kb and contains seven exons.