Figure 2

Distinct Pax6 expression levels among progenitors and postmitotic neurons. Immunocytochemical and flow cytometric analyses of neurogenic retinas. (A) Merged confocal image of PCNA (red) and Pax6 (green) co-labeled E6.5 (HH stage 30) retina. (B) Flow cytometry profile of E6 retinal cells based on PCNA signal and DNA content. The positions of 2n and 4n DNA contents indicate cells residing in the G1/G0 (2n) or G2/M (4n) phase. S phase cells are positioned between 2n and 4n along the DNA content axis. PCNA⁺ cells (boxed, 83.2% of total cells) are distributed throughout the cell cycle. (C) Flow cytometry profiles for Pax6 levels of total cells (left panel) and Pax6 versus DNA content (right panel) at E7 (HH stage 32). Blue lines delineate Pax6^Hi (20.9%), Pax6^Lo (46.5%), and Pax6^Neg (32.6%) cell popuations. (D-G) Merged confocal images of E6.5 (HH stage 30) retinas co-labeled for Pax6 and NF145 (D), Brn3a (E), AP2α (F), or Visinin (G). (D', D") Higher magnification images of NF145 and Pax6 co-labeling near the ventricular surface. Arrows indicate co-labeled cells. Arrowheads point to marker-positive but Pax6-negative cells. (H-K) Flow cytometry profiles of Pax6 and NF68 (H), Brn3a (I), AP2α (J), and Visinin (K) at E7 (HH stage 32). Boxed regions delineate distribution of neuronal marker-positive cells according to Pax6 levels. Percentages of marker-positive cells among total cells are shown above the boxes. gcl, ganglion cell layer; vs, ventricular surface; vz, ventricular zone. Scale bars: 100 μm in (A, G), which applies to (D-G); 50 μm in (D'), which applies to (D', D").