Developmental expression of muscarinic acetylcholine receptors in chick retina: selective induction of M2 muscarinic receptor expression in ovo by a factor secreted by muller glial cells

Abstract

Muscarinic acetylcholine receptors (mAChRs) play an important role in signal processing in the retina. We have used subtype-specific antibodies to identify the changes in the localization of mAChR expression during embryonic development of the retina in vivo and their relationship to the changes in mAChRs in retinal cells in culture. We have demonstrated previously that treatment of fresh retinal cultures with conditioned media from mature retinal cultures specifically induces expression of the M(2) mAChR (McKinnon et al., 1998). We show that the M(2)-inducing activity, which we tentatively have called MARIA (muscarinic acetylcholine receptor-inducing activity) is produced by Müller glial cells in culture, because significant activity can be found in media conditioned by essentially neuron-free cultures of Müller glia, as well as by a Müller glial cell line but not several neuroblastoma cell lines. We also demonstrate that the appearance of the M(2) receptor in vivo occurs concomitantly with the appearance of significant numbers of Müller glial cells in the developing retina. Furthermore, the administration of crude or partially purified preparations of MARIA to developing chick embryos in ovo induces precocious expression of M(2) mAChRs in the appropriate cell types in the retina. These results show that a factor secreted by cultured retinal Müller glia can regulate M(2) mAChR expression in vivo and in vitro and suggest that the secretion of MARIA by Müller glia in vivo may be responsible for the normal induction of M(2) mAChR expression during embryonic development.

Fig. 1.

Colocalization of M₄ mAChRs with markers for retinal neurons and Müller glia. Embryonic retinas from E9, E10, E12, E15, E17, and E19 chicks were sectioned and prepared for immunocytochemistry as described in Materials and Methods. mAChRs were immunolabeled with FITC-conjugated secondary antibody (green), and retinal cell markers were immunolabeled with Alexa 568-conjugated secondary antibodies (red). See Results for specificity of marker antigens. Photographs were taken using confocal microscopy.

Fig. 2.

Colocalization of M₃ mAChRs with markers for retinal neurons and Müller glia. Embryonic retinas from E9, E10, E12, E15, E17, and E19 chicks were sectioned and prepared for immunocytochemistry as described in Materials and Methods. mAChRs were immunolabeled with FITC-conjugated secondary antibody (green), and retinal cell markers were immunolabeled with Alexa 568-conjugated secondary antibodies (red). See Results for specificity of marker antigens. Photographs were taken using confocal microscopy.

Fig. 3.

Colocalization of M₂ mAChRs with markers for retinal neurons and Müller glia. Embryonic retinas from E9, E10, E12, E15, E17, and E19 chicks were sectioned and prepared for immunocytochemistry as described in Materials and Methods. mAChRs were immunolabeled with FITC-conjugated secondary antibody (green), and retinal cell markers were immunolabeled with Alexa 568-conjugated secondary antibodies (red). See Results for specificity of marker antigens. Photographs were taken using confocal microscopy.

Fig. 4.

Application of conditioned medium containing MARIA stimulates transcription of the M₂ mAChR gene. Embryonic chick retinal cultures were prepared from E8 retina, plated in 24-well plates, and transfected on culture day 1 with the M₂promoter–luciferase reporter gene construct, as described in Materials and Methods. After transfection, cells were treated for 24 hr with conditioned media, a partially purified preparation of MARIA, or control medium (see Materials and Methods). The data represent luciferase activity/β-galactosidase activity, expressed as fold-increase compared with controls. Values are the mean ± SEM;n = 3.

Fig. 5.

A, Application of conditioned medium containing MARIA stimulates precocious expression of M₂ mAChRs. Colocalization of M₂ mAChRs with tenascin, a protein found in amacrine cells. Embryonic retinal sections were prepared from E9 chicks that had been exposed to 1.5 ml of the following: CM (injected at E6); partially purified MARIA (for purification procedure, see Materials and Methods, injected at E8); Control, control serum-free media, injected at E6 or E8. At E9, the retinas were removed and prepared for immunocytochemistry as described in Materials and Methods. mAChRs were immunolabeled with FITC-conjugated secondary antibody (green), and tenascin was immunolabeled with Alexa 568-conjugated secondary antibodies (red).B, Application of partially purified MARIA has no effect on expression of M₃ or M₄ mAChRs, nor does it affect expression of cellular markers. Embryonic retinal sections were prepared from E9 chicks that had been treated at E8 with 1.5 ml of flow through that was collected from a DEAE Sephacel column over which conditioned medium that had been concentrated threefold was passed (see Materials and Methods). mAChRs were immunolabeled with FITC-conjugated secondary antibody (green), and cellular markers were immunolabeled with Alexa 568-conjugated secondary antibodies (red). See Materials and Methods for specificity of marker antigens. Photographs were taken using confocal microscopy.

Fig. 6.

MARIA is found in media conditioned by retinal cultures that are essentially neuron-free. A, Immunocytochemistry demonstrating the presence of neuronal cellular markers in retinal cell cultures. Embryonic retinal cultures were prepared from E9 retina and grown on 150 mm plates for 6, 13, 20, or 27 d. Cells were prepared for immunocytochemistry as described in Materials and Methods. Retinal cell markers were immunolabeled with Alexa 568-conjugated secondary antibodies. See Materials and Methods for specificity of marker antigens. All photographs were taken at low power using confocal microscopy. B, Embryonic chick retinal cultures were prepared from E8 retina, plated in 24-well plates, and transfected on culture day 1 with the M₂promoter–luciferase reporter gene construct, as described in Materials and Methods. After transfection, cells were treated for 24 hr with conditioned medium collected on culture day 6 (CD6), culture day 13 (CD13), culture day 20 (CD20), or culture day 27 (CD27). C, Embryonic chick retinal cultures were prepared from E8 retina, plated in 24-well plates, and transfected on culture day 1 with the M₂promoter–luciferase reporter gene construct, as described in Materials and Methods. After transfection, cells were treated for 48 hr with concentrated conditioned medium (CM) collected from a rat Müller glial cell line (MGCL), from SN56 neuroblastoma cells (SN56), and from IMR32 neuroblastoma cells (IMR32). The data represent luciferase activity/β-galactosidase activity, expressed as fold-increase compared with controls. Values are the mean ± SEM;n = 3.