Figure 6

Cells in CE neurospheres cells express rod photoreceptor-specific regulatory and marker proteins. After CE neurospheres were cultured in the presence of PN1CM for 20 days, a small subset of cells was positive of GFP fluorescence, indicating the activation of Nrl promoter (A). A selected field shows three GFP-positive cells immunoreactive to GFP antibody demonstrating the specificity of the Nrl-GFP-fluorescence, similar to that in the section of PN1 Nrl-GFP mouse retina. That the rare Nrl-GFP-positive cells were of rod photoreceptor lineage was demonstrated by co-localization of rhodopsin immunoreactivities, detected by RetP1 (upper panel) and Rho4D2 (lower panel) with Nrl-GFP fluorescence, as in PN1 Nrl-GFP retinal sections (B). The proportion of cells with rhodopsin immunoreactivities, detected by RetP1 and Rho4D2, was significantly higher in cells in differentiation conditions than in controls (C). Western analysis of cells after 20 days of differentiation revealed 40 kD and 70 kD bands, immunoreactive to Rhodopsin and Rhodopsin Kinase (RK), respectively (D). Examination of species-specific difference in the retinal potential of CE cell revealed mouse and rat CE neurospheres, subjected to identical culture in PN1CM, generate 4.93% and 12.8% of Rho4D2 positive photoreceptors, respectively, on FACS analysis (E). Calcium imaging by Fura2 revealed the mobilization of intracellular calcium by differentiated cells in the presence of agonist DCPG (DCPG+) and not in its absence (DCPG-), demonstrating the presence of mGluR8 metabotropic glutamate receptor, expressed by rod photoreceptors in vivo (F). Controls: CE untreated cells. Bar illustrates fluorescence intensity in a pseudo-color scale. ONL = outer nuclear layer; INL = inner nuclear layer. Scale = 50 μm.