Figure 5

Increased numbers of Nr2e1 ^frc/frc amacrine cells were not rescued in P7 Wt↔frc chimeras. Chimeric eyes were immunostained for the pan-amacrine marker Pax-6. a In Wt↔Wt chimeras, the density of EGFP positive (green) amacrine cells appeared similar to the density of EGFP negative amacrine cells. In Wt↔frc chimeras, the density of amacrine cells that were EGFP positive (green) appeared higher than the density of EGFP negative amacrine cells. Representative images of a 29 % Wt↔Wt and a 58 % Wt↔frc chimeric retina are shown. The arrow shows a region with high numbers of mutant cells (EGFP positive) where amacrine cells (Pax-6 positive) are overrepresented. b,c Quantification of the density of amacrine cells that were derived from host blastocyst or ESCs in chimeras was assessed by counting single-labeled cells (Pax-6 positive and EGFP negative) or double-labeled cells (Pax-6 positive and EGFP positive) and dividing them by the EGFP negative or EGFP positive retinal area (ONL + INL), respectively. Cell counts were performed in both the INL and the GCL. In the INL, large and round nuclei adjacent to the OPL, presumably belonging to horizontal cells, were excluded from counting. Amacrine numbers in the GCL were obtained by subtracting Brn3a positive ganglion cells from the total Pax-6 positive cells in this layer. In Wt↔frc chimeras, the density of Nr2e1 ^frc/frc amacrine cells (EGFP positive) was (b) higher (49 % increase) than the density of wild-type amacrine cells in the INL but was (c) similar to wild type in the GCL. n = 3 for Wt↔Wt, n = 3 for Wt↔frc; *, P ≤ 0.05; ns, not significant; error bars represent SEM; GCL, ganglion cell layer; Hoechst, nuclear counterstain (blue); INL, inner nuclear layer; neg., negative; pos., positive; scale bar = 50 μm