concentrated on medaka [6]. The fact that the MBT of both species starts at different time points gives a first hint that data on zebrafish MBT might not be representative for all fish species and that a closer look on medaka embryos is justified. Unexpectedly, we find that a large number of medaka embryos show highly asymmetric cell divisions as early as from the second cell division onwards. This asymmetry is directly reflected in different cell sizes and cytoplasmic volumes, but surprisingly without negative effects on normal development. Medaka embryos also lose cell-cycle synchrony already at cleavage 5, leading to transition from synchronous to metasynchronous cell division. Most surprisingly, we observed strong RNA polymerase II phosphorylation down from the 64-cell stage and the subsequent initiation of mRNA transcription. This might be connected to regulatory processes that prepare embryos for the MBT or even mark the beginning of MBT itself at a much earlier time point than has been reported previously. Results Asynchronous cell divisions in early embryos The cell cycle during cleavage stages is generally described as being short and highly synchronous. Lengthening of the cell cycle and upcoming asynchrony is taken as a sign for the beginning MBT [1]. For Zebrafish, this event has been mapped to cycle 10 [2]. Time-lapse investigations in our laboratory on medaka fish provided preliminary evidence that embryos from this species lose cell division synchrony several stages before MBT occurs in zebrafish, and establish a cell division behavior that is called ‘‘metasynchronous cell division’’ (Movie S1). In order to investigate this phenomenon further, confocal imaging was used to visualize differences in cell synchrony at defined stages. We found that cell division is synchronous in embryos until cell division 4, when embryos progress from 8 to 16 cells. First indications for an upcoming asynchrony appear at cell division 5, 16 to 32 cells, when a temporal spacing of the cell cycle between single cells can be detected (Fig. 1A). During the following division cycles, cell division synchrony in embryos is not lost completely. Rather, it is replaced by a specific division pattern that is called ‘‘metasynchrony’’, which is defined by cell division progressing in waves that start in the embryos’ center and spread out to their periphery. In early medaka embryos, cells are usually arranged in an elongated or rectangular manner until the 16-cell stage and in a roundish, disc-like manner from the 32-cell stage on until gastrulation. Starting at cycle 6 (32 to 64 cells), when embryos have a sufficient number of cells to form well-defined central and peripheral regions, more and more embryos (5/13) are found in which a temporal spacing of mitosis initiation between central and peripheral cells can be identified (Fig. 1A, B). This temporal spacing of mitosis initiation increases during the following divisions. At the latest with division 8 (128 to 256 cells) it reaches an extent at which all central cells have already re-entered interphase while peripheral cells are still in late anaphase or telophase (Fig.1B, Fig. S1). These findings were confirmed by time-lapse observations of embryos that were fluorescently tagged by injection of mRNA for an eGFP labeled Histone2B protein (H2B-eGFP) and imaged throughout the cleavage phase. These embryos showed highly synchronous cell divisions until the 16-cell stage and an emerging minor temporal spacing during the division from 16 cells to 32 cells (Fig. 2). During the following divisions, this temporal spacing again increased until cell division initiation could be clearly detected first in centrally located cells and later in peripheral cells. Asynchronous Cell Division in Medaka before MBT For example, karyokinesis at division 7 (64 to 128 cells) has finished in central cells while peripheral cells are still at late anaphase or telophase (Fig. S2, Movie S2). Tracking the size (as a determined area) of a single cell’s nucleus and its daughter cells from a mid 8-cell stage to the late 64-cell stage at constant time intervals for 45 consecutive measurement points illustrates the relationship between the metasynchronous cell cycle and the position of a cell within the embryo. Nuclei have their largest area during interphase and this value decreases in preparation for the next mitosis. The nuclear signal reaches its smallest area at the metaphase when daughter chromosomes are separated. Comparing the 4 displayed daughter cells at the passage from 32 to 64 cells, the most central cell has condensed its chromatin one measurement point earlier than the other 3 cells. Later, the daughter cells of this early cell have entered cell division two measurement points before the others (Fig. S3). Furthermore, some of the embryos that were observed during the time lapse experiments, divided asymmetrically from the 2-cell to the 4-cell stage. In these embryos cell division also de- synchronized at the cycle from 16 to 32 cells, but at the following stages not each one of these embryos did develop the pattern typical for the symmetrically dividing embryos described above, where central cells cycled first and peripheral cells later (Fig. S4). In the given example, cells first started division at one pole of the embryo and cells on the opposite pole followed later (Fig. S5, Movie S3). Asymmetric cleavage at the 2-cell stage does not affect embryo development Time-lapse observations of injected medaka embryos showed the occurrence of highly asymmetric cleavages from the 2-cell to the 4-cell stage. For a detailed analysis the cleavage furrows and the cell-arrangements of medaka embryos at the 4-cell stage were investigated and consequently three distinct classes of embryos were defined. They were designated type I, type II and type III (Fig. 3). Embryos with almost perfect 90u cleavage furrows in the center of the four cells and symmetry along the X and Y axes were classified as type I embryos (Fig. 3Ai, Aii). Generally, these embryos have four homomorphic cells, yet we occasionally observed that two of the four cells were seemingly smaller than the others. Type II embryos still feature 90u cleavage furrows but have lost axial symmetry (Fig. 3Bi, Bii). The interception point of the cleavage furrows is located at the center of the four cells, but the furrows no longer build a cross-like structure. In a type I embryo, cells only have contact to their directly neighboring cells, but in a type II embryo two cells now also have contact to their opposing cell, forming an hourglass-like structure. Type III embryos include all the embryos that lack any symmetric cleavage furrow or clear organization of the cells or both (Fig. 3Ci, Cii; Fig. S6). To investigate if asymmetric cleavages have a negative effect on embryonic development, the survival rate of the three different cleavage types was determined. Altogether, 774 embryos were classified according to their cleavage furrows: 171 belonged to type I (22%), 424 to type II (55%) and 179 to type III (23%) (Fig. 3D). These embryos were raised under standard conditions and monitored for early embryonic death. Surprisingly, among the 774 embryos, only one died before hatching and this one was scored as type II. The remaining embryos developed normally and hatched on time around day 7 post fertilization. Time-lapse observations of developing embryos from the 4-cell stage up to the 1024-cell stage were performed to investigate possible effects of the cleavage type on development. Type I embryos developed just like the idealized medaka embryo as PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e21741