Figure 2. Effects of forced expression of M6a on retinal differentiation and proliferation. A: Sublayer distributions of virus-transduced enhanced green fluorescent protein (EGFP)-positive cells in retinal explants. Retinal explants were infected with retorovirus particles that encode M6a and EGFP. After 14 days, the explants were harvested and frozen-sections prepared. Immunostaining was performed using an anti-EGFP antibody. The percentages of cells in each sublayer are shown. More than 200 cells were counted for each sample, and the standard deviation (SD) was calculated from three independent experiments. B: Differentiation of virus-infected cells examined by immunostaining to identify subpopulations within the retina. The percentages of marker-positive cells in the EGFP-positive population are shown. Rhodopsin for rod, HuC/HuD for amacrine, glutamine synthetase for Müller glia, and protein kinase C for bipolar were used as markers. More than 100 cells were examined for each sample, and the average value from three independent experiments is shown with the SD. C,D: Proliferation of M6a-expressing retinal cells was examined by measuring incorporation of bromodeoxyuridine (BrdU; C) or expression of the Ki67 antigen (D). BrdU was present for the final 24 h of four days of culture of retinal explants, and frozen sections were immunostained using antibodies against BrdU. The same samples were immunostained with the anti-Ki67antibody. The percentage of positive cells with SD are shown. Listed below each panel is the stage when each retinal explant was prepared and its culture period. The following abbreviations are in effect: outer nuclear layer (ONL); inner nuclear layer (INL); ganglion cell layer (GCL).