To complement the above analyses, we wanted to perform kinetic studies to define when markers are induced relative to the end of S-phase and G2/M. Prior work defined the average length of S-phase and the entire cell cycle at various times during rat retinal development (Alexiades and Cepko, 1996). If the length of G2/M was known, BrdU-labeling could be used to define when markers are extinguished or induced relative to cell birth.
G2/M length is determined by measuring the time necessary to BrdU-label all M-phase cells (here defined by PH3 staining), which is the percentage of labeled mitosis (PLM) method (Quastler and Sherman, 1959). Varying lengths of BrdU chase are used to determine how fast progenitors at the end of S-phase, and thus labeled with the BrdU pulse, reach the end of M-phase. When the chase period after BrdU-labeling is too short, cells that were at the end of S-phase do not reach the end of M-phase and thus some PH3+ cells remain BrdU−, but when the chase period is as long as G2/M all PH3+ cells become BrdU+. We observed that in the post-natal retina, it took no less than 30 min post-BrdU injection for cells in the NBL to accumultae enough BrdU to ensure a reproducible detection and quantification. Thus, we considered the start of the chase to be 30 min after BrdU injection at all developmental time points examined. For example, if retinas were harvested 30 or 60 min after the BrdU injection, the time of chase would be 0 or 30 min, respectively. We assessed six chase periods (30, 50, 70, 90, 110, and 130 min) at three embryonic time points (E12.5, 14.5, and 16.5), and eight chase periods (30, 90, 110, 130, 150, 170, 190, and 210 min) at three post-natal time points (P0, P3, and P5). In the embryonic retina at least 90% mitotic cells were BrdU+ after a 70-min chase, which lengthened to 110 min in the post-natal retina (Fig. 6A). In line with live imaging studies indicating that M-phase is ∼30 min (Cayouette et al., 2003; Gomes et al., 2011), most PH3+ cells became BrdU+ in a single 20-min chase window (Fig. 6A, Table 7). However, the slope of the labeling curve then flattened and there was a considerable lag between the chase time required to double-label the first ∼90% versus the last ∼10% of cells, which lengthened across development; 20 min/E12.5, 40 min/E14.5, 60 min/E16.5, and ∼100 min post-natally (Fig. 6A, Table 7). These data suggest that a small fraction of progenitors (≤10%) take longer to complete G2 than the bulk of the population. To validate the BrdU/PH3 analyses, we also assessed the fraction of cells that exhibited perinuclear Cyclin B2, which marks late G2. As expected, the fraction of B2+ cells that were BrdU+ slightly exceeded that of PH3+ cells at each time point assessed (Fig. 6B, Table 8). The BrdU chase-PH3 analyses reveal the typical and maximum length of G2/M (Tables 7, 9), and the latter is critical to assess unambiguously whether cell-cycle proteins are detectable after cell birth or, similarly, whether neural markers are induced only after cell birth.