Figure 4

Divergence of Pax6 protein expression in the G2/M phase of the cell cycle. (A) Flow cytometric analyses of BrdU, Pax6, and DNA content among E6.5 (HH stage 30) retinal cells after a 20-minute BrdU pulse labeling. Cell density profiles of BrdU⁺ (upper row) and BrdU^- (lower row) cells at various post-chase times are shown. The 0 hour point represents the time when cells are dissociated after the pulse label and then fixed and processed for flow cytometry. Each data point represents a minimum of 30,000 cells analyzed. The x-axis represents DNA content for G0/G1 (2n), S (between 2n and 4n), and G2/M (4n) phase cells. (B) Merged flow cytometry profiles for BrdU⁺ (green) and BrdU^- (red) cells at 0, 3, and 6 hours post-BrdU pulse labeling. The purple frames subdivide G2/M phase cells (4n) according to Pax6 levels into high (Hi), low (Lo), and negative (Neg) categories. Between 0 and 6 hours of chasing, Pax6^Hi cells clearly increased, demonstrating the Pax6 level divergence when cells enter the G2/M phase. (C) Quantification of Pax6 levels at stage 30 (E6.5) and stage 33/34 (E7.5 to 8) among BrdU⁺ cells that had entered G2/M phase (4n) as shown in the three boxed regions in (B). The percentages of 4n BrdU⁺ cells with Pax6^Hi, Pax6^Lo, or Pax6^Neg levels among total 4n BrdU⁺ cells at various post-chase times are shown (N = 3; mean ± standard error). Asterisks represent P-values between 0 hours and various chase time points: *P < 0.05; **P < 0.01; ***P < 0.001.