Maturation Of Gs Inducibility In Embryonic Chick Neural Embryo

R5 R6 R7 R8 R® R10 R" R12 R14 EMBRYONIC AGE AT EXPL ANTATION-24 hr CULTURES ±HC

Figure 2.16. Glutamine synthetase induction in developing chick retina. Development of inducibility for glutamine synthetase in chick retina between 5 and 14 days of incubation; relation to embryonic age and to changes in cell number and in total protein per retina. Retinas dissected from embryos were cultured for 24 hr in medium with cortisol (+H) or without it (-HC). The black bars show the levels of GS activity induced in retina of different embryonic ages. The white bars show the levels of GS in the absence of the steroid inducer (Moscona and Moscona, 1979). (Copyright 1979 Springer-Verlag New York, Inc., reprinted with permission.)

R5 R6 R7 R8 R® R10 R" R12 R14 EMBRYONIC AGE AT EXPL ANTATION-24 hr CULTURES ±HC

Figure 2.16. Glutamine synthetase induction in developing chick retina. Development of inducibility for glutamine synthetase in chick retina between 5 and 14 days of incubation; relation to embryonic age and to changes in cell number and in total protein per retina. Retinas dissected from embryos were cultured for 24 hr in medium with cortisol (+H) or without it (-HC). The black bars show the levels of GS activity induced in retina of different embryonic ages. The white bars show the levels of GS in the absence of the steroid inducer (Moscona and Moscona, 1979). (Copyright 1979 Springer-Verlag New York, Inc., reprinted with permission.)

Figure 2.17. Glucocorticoid receptor expression changes in developing retina. A. Immuno-blotting studies with GR in E6 and E10 retinas. The blots show nuclear and cytoplasmic distribution of GR isoforms in early and middevelopmental ages. B. The histogram shows differential levels of the GR isoforms in E6 and E10 retinas. C. Immunocytochemical localization of GR in developing chick retina. E6 (A,C) and E12 (C,D). A and C are paraffin sections, and B and D are cryostat sections stained with glucocorticoid receptor antibody. The immuno-staining is present in all cells initially but becomes restricted to a small group of cells (and their nuclei) as the retina matures (Gorovits et al., 1994). (Copyright 1994 National Academy of Sciences, U.S.A., reprinted with permission.)

Figure 2.17. Glucocorticoid receptor expression changes in developing retina. A. Immuno-blotting studies with GR in E6 and E10 retinas. The blots show nuclear and cytoplasmic distribution of GR isoforms in early and middevelopmental ages. B. The histogram shows differential levels of the GR isoforms in E6 and E10 retinas. C. Immunocytochemical localization of GR in developing chick retina. E6 (A,C) and E12 (C,D). A and C are paraffin sections, and B and D are cryostat sections stained with glucocorticoid receptor antibody. The immuno-staining is present in all cells initially but becomes restricted to a small group of cells (and their nuclei) as the retina matures (Gorovits et al., 1994). (Copyright 1994 National Academy of Sciences, U.S.A., reprinted with permission.)

are present in the retina before day 15, but the lack of circulating glucocorticoids prevents induction of glutamine synthetase. Moreover, glutamine synthetase induction in this system appears to accompany Müller cell differentiation.

Although the molecular mechanisms underlying glutamine synthetase induction are complex and not completely understood, experimental evidence suggests that Müller cell-specific expression is achieved through the concerted involvement of positive and negative cis elements, a glucocorticoid response element (GRE) and a neural-restrictive silencer (Zhang et al., 1993; Li et al., 1996; Avisar et al., 1999). In addition, it is quite clear that glucocorticoid receptors play an important role in this process (Zhang et al., 1993; Grossman et al., 1994; Gorovits et al., 1994). Results from a recent developmental study indicate that glutamine synthetase expression in chick retina is strongly dependent on the level of a 95 kDa glucocorticoid receptor (Gorovits et al., 1994). At early developmental stages, all retinal cells express the receptor, and consequently all cells express glutamine synthetase. At later developmental stages, however, receptor expression appears to be lost from all retinal cells except Müller cells. As a consequence, glutamine syn-thetase can be induced only in Müller cells (Fig. 2.17). Interestingly, transcription of the glucocorticoid receptor gene is strongly repressed by the c-Jun protein, which is expressed in proliferating neuroblasts (Vardimon et al., 1999).

Whole Ceil Monolayer Retina Aggregates Cultures

Whole Ceil Monolayer Retina Aggregates Cultures

Figure 2.18. Glutamate synthetase induction is dependent on cell contact. Northern blots show that GS mRNA induction is higher in cell aggregates and retina than in monolayer cultures of Müller cells. Note that CAII and H3.3 levels do not change appreciably. The figure to the right shows a proposed model for glutamine synthetase induction in Müller cells (GLIA) (Moscona and Vardimon, 1988). (Copyright 1988 Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc., reprinted with permission.)

Figure 2.18. Glutamate synthetase induction is dependent on cell contact. Northern blots show that GS mRNA induction is higher in cell aggregates and retina than in monolayer cultures of Müller cells. Note that CAII and H3.3 levels do not change appreciably. The figure to the right shows a proposed model for glutamine synthetase induction in Müller cells (GLIA) (Moscona and Vardimon, 1988). (Copyright 1988 Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc., reprinted with permission.)

A Müller Cell

Growth _ Factors

CAMs

ECM GFs

Proliferation

Migration

Differentiation

Proliferation

Migration

Differentiation

B Neuron

Mitosis Inhibition

Gene

Regulation

Figure 2.19. Potential Müller cell-neuron interactions in developing retina. A. Influence of Müller cells on the proliferation, migration, and differentiation of neurons. These processes are mediated by growth factors, cell adhesion molecules, and extracellular matrix molecules; B. Neuronal influence on Müller cell activity. Neurons may regulate the mitosis and gene activity in glial cells.

B Neuron

Figure 2.19. Potential Müller cell-neuron interactions in developing retina. A. Influence of Müller cells on the proliferation, migration, and differentiation of neurons. These processes are mediated by growth factors, cell adhesion molecules, and extracellular matrix molecules; B. Neuronal influence on Müller cell activity. Neurons may regulate the mitosis and gene activity in glial cells.

Perhaps the most remarkable feature of the system is that contact interaction with neurons appears to be essential for glutamine synthetase induction in Müller cells. When retinal cultures containing both neurons and Müller cells are treated with glucocorticoids (Cortisol), glutamine synthetase is strongly induced in Müller cells (Moscona, 1983). However, if Müller cells are grown in monolayer cultures in the absence of neurons, hormone treatment fails to induce glutamine synthetase (Fig. 2.18). Although the mechanism underlying this phenomenon is still not established, it appears that disruption of neuron-glia contact leads to activation of the c-Jun signaling pathway which in turn inhibits transcription of glucocorticoid receptor gene (Reisfeld and Vardimon, Vardimon et al., 1999). In the absence of the receptor, glutamine synthetase induction becomes non-responsive to glucocorticoids.

Neuronal interaction is apparently not a universal requirement for gene expression in Müller cells (Fig. 2.19). Although glutamine synthetase induction is dependent on neuronal contact, synthesis of CA II, filamin, or CRALBP is not affected by the absence of neurons (Linser and Moscona, 1981; Hicks and Courtois, 1986; Lewis et al., 1988; Lemmon, 1986; Sarthy et al., 1998; Roque et al., 1997). Clearly, the expression of many Müller cell-specific proteins does not require contact with neurons.

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