Asynchronous Cell Division in Medaka before MBT Figure 3. Asymmetric cell divisions at cycle 2. (Ai, Bi, Ci) Characterization of three types of medaka embryos at the 4-cell stage with regard to the grade of axial symmetry of the cell cleavages. (Ai) Type I embryo showing highly symmetric cleavages and right-angled cells. (Aii) Cell boundaries were highlighted to help visualization. (Bi; Bii) Type II embryo showing slight but clearly detectable impairment of axial symmetry, while still having right-angled cell shapes at the interception point. (Ci; Cii) Type III embryos lacking axial symmetry. (D) Frequency of the three embryo types at 4-cell stage medaka embryos among 774 eggs. Type I occurred with a frequency of 22% (171/774) of total embryos (green bar), type III embryos with 23% (179/ 774) (orange bar). Type II embryos represent the largest group of the three types with 55% (424/774) of all embryos (blue bar). doi:10.1371/journal.pone.0021741.g003 Interestingly, this metasynchronous division pattern is distorted in embryos with asymmetric early cleavage furrows. Usually, embryos during cleavage phase show several levels of axial symmetry. At the 4-cell stage, cells are oriented in a clover leave like configuration, followed by a double row of 264 cells at the 8- cell stage, the 464 ‘‘chessboard’’ at the 16-cell stage and finally the upcoming roundish, disk-like arrangement at 32 cells and later stages. Embryos not showing this high level of symmetry are also more likely to be unable to establish a clear metasynchronous cell division, although they still establish a certain level of structured and organized division pattern. However, the regulatory mecha- nism behind this behavior remains unclear. Asymmetric cell cleavages and unequal cell volumes at the 4-cell stage Asymmetric and unequal cell divisions occur in medaka already at the 4-cell stage. These asymmetric divisions produce divergent shapes of embryos instead of embryos consisting of cells that are even in shape and volume like a symmetric cell division would produce, and like it was described for medaka by Iwamatsu [20]. Our study demonstrates that only about one quarter of embryos follow the scheme of the idealized embryo with highly symmetric cell divisions. More or less strong deviations of the symmetric divisions represent the majority of possible cleavages and another quarter of embryos show extremely asymmetric cleavages. However, the deviation from symmetry has no influence on the further course of embryonic development. Unequal cell cleavages have already been reported for lower animals like leech [22], Caenorhabditis elegans [23] and sea urchin [24], but to our knowledge never before for a vertebrate. MBT in general is regarded to be regulated by the nucleo- cytoplasmic ratio [1][2][9]. Consequently, unequal cell volumes, if differences are big enough, should cause alterations in MBT onset [3][5]. Unexpectedly, as our study shows, already cells at the 4-cell stage in medaka embryos can differ in volumes over a large scale. The inequality of cell volume directly reflects the grade of asymmetric cell cleavage. In zebrafish it has been reported that the cell cycle becomes asynchronous at cleavage 9, and it was suggested that this asynchrony is due to volume differences that may result from unequal early cell divisions [2]. However, this study did not provide cell volume data prior to cycle 9. We speculate that unequal cell volumes at cycle 9 in zebrafish are just the consequence of early asymmetric cell divisions and early unequal cell volumes like it is the case for early medaka. Early RNA polymerase II transcriptional activity Phosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII) is associated with active RNA transcription [25][26][27][28]. In medaka, phosphorylation was detected first during division from 8 to 16 cells, but phosphory- lation stayed rare and sporadic until the 16-cell and 32-cell stages. Consistent phosphorylation of RNAPII in a large fraction of cells is established only by the 64-cell stage, even though cells at the periphery of the embryo usually do not show any phosphorylation until early blastula stage. A lack of phosphorylation was noted in more centrally located cells of some embryos at the 128-cell stage. RNAPII is hypopho- sphorylated during mitosis [29] as transcription is repressed at this process [30]. In consequence, this could indicate that the cell cycle in embryos of this stage becomes metasynchronous as cells are at different steps of the cell cycle. This pattern is blurred in later stages, when phosphorylation-positive and negative cells are strongly mixed together and the phosphorylation pattern develops a more mosaic character. This change is caused by the increase in cell asynchrony during the embryos transition towards early blastula and the beginning MBT. Importantly, until now only one study attempted to identify the time point of MBT activation in medaka by investigating the first appearances of paternal transcripts by RT-PCR. This study determined the beginning of MBT in medaka at around stage 11 (2000 to 4000cells) [6]. In contrast, our study proves that RNA polymerase II gets phosphorylated in a small fraction of cells already at the 16-/32-cell stages and strongly in larger fractions by the 64-cell stage. Furthermore, our findings also demonstrate that transcription in medaka embryos is detectable at earlier time points (between 64 to 1000 cells) than it was assumed so far. Previous microarray analysis on mRNA levels of over 16.000 genes from zebrafish embryos have shown that mRNA levels of 125 genes are increasing at the 64-cell stage. Many of these genes are involved in protein degradation, cell proliferation, cell growth, cell adhesion and RNA synthesis and stability. It was supposed that these genes need to commence their function during a pre- MBT state to ensure full functionality during MBT and following embryonic development. The data reported here for early medaka embryo development conflict with the general descriptions for fish embryos before the midblastula transition, namely being synchronous, with equal cell division and no transcription occurring before MBT, which was set to stages beyond 512 cells. Still, our results do not contradict the previously postulated mechanisms for MBT onset. Chromatin regulation, maternal clock, transcript abortion and nucleo-cytoplasmic ratio are all mechanistically compatible with our observations. The here reported unequal distribution of cytoplasm, the wave- like cell cycle pattern together with very early RNAPII-activity and the initiation of transcription before MBT do, however, demon- strate that early development of medaka is more robust and less strictly controlled than was expected. Furthermore, our measure- ments imply that MBT is not an all-or-nothing, digitally-switched process, but rather a progressive event that is independently occurring in individual cells. Materials and Methods Ethic statement This research did neither involve human participants nor any human material. All experiments involving live animals were conducted in accordance with the German Animal Protection Law. Our laboratory and animal facilities are approved for such work and are regularly inspected and supervised by the animal protection officers of the University of Wu¨ rzburg and the Government of Lower Franconia. OLAW: A-5864-01. PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e21741