Asynchronous Cell Division in Medaka before MBT Figure 1. Desynchronization of cell cycle in early medaka embryos. Progression of cell cycle desynchronization in early medaka embryos before MBT. (A) Image shows staining for DNA at 4 different cell divisions (4–8 cells, 8–16 cells, 16–32 cells, 32–64cells). (Ai–Aii) Synchronous cell division at cycle 4 to 8 cells and at cycle 8 to 16 cells. (Aiii–Aiiii) Asynchronous cell division at cycle 16 to 32 cells and at cycle 32 to 64 cells. (Aiii) Asynchronous cells at cleavage from 16 cells to 32 cells at random positions in the embryo; nuclei are at interphase (yellow arrows) or at late mitotic phase (green arrows). (Aiiii) Asynchronous cleavage from 32 to 64 cells, cells in the center are in late mitotic phase (green arrows), most cells at the rim are at interphase (yellow arrows). (B) Progression from synchronous to asynchronous and from asynchronous to metasynchronous cell division between cycle 2 (2–4 cells) and cycle 9 (256–512 cells). Embryos that divided synchronously are represented by yellow bars, embryos that divided asynchronously by green bars and metasynchronously dividing embryos by blue bars. Note that more synchronous dividing embryos are found during the first three cell divisions (2–4 cells, 4–8 cells , 8–16 cells) than during the second three cell divisions (16–32 cells, 32–64 cells, 64–128 cells) (Chi-test with p,0.001; for this, metasynchronous divisions were also counted as asynchronous divisions). Later, the number of embryos showing a random asynchronous division pattern drops and more and more embryos show a clear metasynchronous cell division. By the division from 64–128 cells, the majority of the division pattern has changed from asynchronous to metasynchronous (p,0.001). doi:10.1371/journal.pone.0021741.g001 described by Iwamatsu [20]. They start with a typical clover leaf shape and stay symmetric until stage 10, 1024 cells, when the embryos reach the typical roundish disc of the early blastula stage (Fig. S7). Type II embryos differ only at early stages from Iwamatsus ideal embryo. At the 8-cell or 16-cell stage, embryos sometimes showed slightly shifted shapes as cells were not arranged in a symmetric manner. These shape differences were usually compensated until the 32-cell or 64-cell stage, respectively. Even type III embryos were able to establish the typical disc of the early blastula stage. However, in these embryos this process required more cell divisions and thus more time depending on the grade of deviation from the type I development (Fig. S8). In the given example, the embryo was not able to fully compensate before 1024 cells (Fig. S9). Unequal cell volumes at the 4-cell stage In order to measure cellular volumes, embryos were stained with CellMask DeepRed. However, this technique is not useful for measuring embryos before the 4-cell stage because the cellular boundaries to the cytoplasm are not apparent enough to clearly define the shape of the cell. Only at the 4-cell stage the cellular border has reached a level of clarity that allows the discrimination of single cells (Fig. 4A). Fluorescent staining and confocal imaging technique was chosen in order to monitor changes in all three dimensions (Fig. S10). A total of 33 embryos were scanned (11 type I, 11 type II and 11 type III) (Fig. 4B, Table S1). Of these embryos showed only very few similar or equal cell volumes (5/33). Many consisted of three cells of similar volume and a relatively large or small fourth cell (20/33). If cell volumes are examined with respect to the three embryo types, it appears that the level of asymmetry is reflected by the level of differing cell volumes. In particular, 9/11 of type I embryos were found within the 16 embryos with the most similar volumes between the largest and the smallest cell. In contrast to that, 11/11 of type III embryos were found within the 17 embryos with the most different volumes between the largest and the smallest cell (Fig.S11). However, there is no strict correlation since some type I embryos were found that differed more in cell volumes than type II embryos as well as some type II differed more than type III (Fig. S12). The fold changes between the largest and the smallest cell of each embryo were calculated (Fig. 4C, Table S1). The smallest PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e21741