Figure 2. Asynchronous cell division appears in living embryos at cycle 5. Maximum intensity collapses of an embryo at 3 different time points of cycle 5 (16 to 32 cells) during time laps observations. (A) Nuclei of interphase cells are large due to the decondensed chromatin. (B) The nuclei in some cells are shrinking when their chromatin starts to condensate (green arrows), while the chromatin of other cells remains decondensed (yellow arrows). (C) Some cells are at ana-/ or telophase (green arrows), while remaining cells only have started to condensate their chromatin (yellow arrows). doi:10.1371/journal.pone.0021741.g002 observed fold change in an embryo was 1.05. Most embryos ranged within a fold difference between 1.3 and 1.5 (15/33), but values up to 2.69 were found as well. Type I embryos range from 1.05 to 1.47 times difference, type II from 1.24 to 1.74 and type III from 1.49 to 2.69 (Table S1, Fig. S13). Asynchronous Cell Division in Medaka before MBT RNA polymerase II phosphorylation and transcriptional activity in early Medaka To monitor the initiation of zygotic transcription, we started with the investigation of RNA polymerase II (RNAPII) phosphor- ylation. Unexpectedly, earliest phosphorylation was already detected in nuclei of a fraction of cells in embryos at the 16-cell stage (Fig. 5A). Positively stained nuclei remained rare during 16- cell and 32-cell stages with 17% of all embryonic cells showing positive staining at the 16-cell stage and 30% at the 32-cell stage respectively. A specific spatial pattern for this time points could not be identified (Fig. 5A, B). By the 64-cell stage, the number of phospho-RNAPII positive cells increased to 73% (Fig S15). Cells that were not stained positive were exclusively located at the periphery and never at central positions (Fig. 5C). At the 128-cell stage, the phosphorylation level was still high (68% of all cells). Peripheral cells were still negative for phospho- RNAPII, but negative cells or cells with very low levels of phosphorylation were now also found at more central positions of the embryo (Fig. 5D). Positive and negative cells were intermingled during the 256- and 512-cell stage (Fig. S14B) and peripheral cells showed polymerase II phosphorylation only after the 1024-cell stage (Fig. S14C). Transcriptional activity at these early stages was verified by RT- PCR. For this, a selection of target genes were investigated for transcriptional up-regulation at stages 0–2 (0–2 cells), 8–10 (64– 1000 cells), 11 (early-late blastula), and stage 14 (pre-mid gastrula). Ccnb1, a member of the AB subfamily of cycline proteins that control the G2/M transition, showed strong up-regulation between stages 8 to 10 and 11. RPS12, which encodes for a member of the 40S ribosomal subunit, showed induction at stage 11 and robust induction at stage 14. PSMC1, a protease, (and four other genes, data not shown), showed no upregulation during the investigated stages (Fig. 6). Discussion Asynchronous and metasynchronous cell division starts between the 16 and 64 cells stages In the classical studies on cell cycle duration in early vertebrate embryos data were obtained from time-lapse observations, which were based on standard light microscopy. We used confocal microscopy to detect earliest differences in cell cycle synchrony. With this technique, it was possible to detect first alterations and asynchrony at cycle 5 (from 16 to 32 cells) when the first cells became apparent that divided prior to the remaining cells. Asynchrony is a direct result of cell cycle lengthening at beginning MBT in Drosophila, Xenopus and zebrafish [2][5][13]. However, the loss of synchrony in medaka embryos at cycle 5 is not connected to a beginning MBT, but marks the beginning of a metasynchronous cell cycle. The cell cycle in zebrafish embryos is rapid and synchronous from 2 to 128 cells. Metasynchronous cell division emerges at cycle 8 (from 128 to 256 cells) [21] and is well established at the early blastula stage at cycle 9 (from 256 to 512 cells) [2]. Although medaka embryos lose overall synchrony already at cycle 5, and start to develop a temporal spacing of mitosis initiation between central and peripheral cells right away. However, embryos at the 16-cell or 32-cell stage do not possess sufficient cells to form a distinct center and periphery or a distinct difference between both areas. This is probably the reason why it takes up to two or three additional cell divisions, until cycle 7 to 8, before embryos first displayed a cell cycle that occurs in clear waves - a typical feature of metasynchronous cell division. PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e21741