The splitting into two individual nuclear signals was considered as breaking point for classifying the cell as being in mitotic phase. This is accompanied by an elongated shape and symmetry along an imaginary plane between both chromosome-sets. Loss of the elongated shape and the clearly detectable symmetry of the nuclear signal were considered to mark the end of the mitotic phase and the re-entry into interphase. Embryos were also classified as asynchronous if all cells indeed showed mitotic character, but are at different time points or progression levels of the metaphase. A close position between the daughter chromosomes represents nuclei that have just entered anaphase, whereas a large distance represents nuclei that are at late ana- or telophase and have entered mitosis prior to the early anaphase cells. Cell volume determination Confocal images were loaded into the Volocity software and filtered. Single cells were isolated by manually performed optical dissection on individual embryos. Only embryos that did not change the shape and X-/Y-position of their cleavage furrow along the Y-axis were used for this procedure. Cell volumes were measured in Volocity with an object-identifying protocol that was adjusted at the beginning and remained unchanged for all cell volume measurements. The volumes of all 4 cell volumes of each embryo were summed up and set as 100%, and the relative volume of each cell was calculated relative to the total volume of the particular embryo. These relative volumes were used for comparisons of cell volumes within individual embryos and between different embryos. Embryos were considered as ‘‘consist- ing of cells with similar volumes‘‘ if the relative volumes’differences between the smallest and the largest cell did not exceed 5%, which is a volume difference between both cells close to or smaller than the factor 1.2 from the smallest to the largest cell. Embryos were considered as ‘‘consisting of three cells of similar volume and an extremely large or small fourth cell’’ if the differences between the relative volumes’differences of the smallest and the largest cells to the embryos mean relative volume did not exceed the factor 2. The embryos mean relative volume was calculated between the 2nd smallest and the 2nd largest cell: {[(B+C)/2]-A}/{D-[(B+C)/ 2]} with A as the smallest and D as the biggest cell. Additional information for the calculations can be found in Table S1. In vitro mRNA transcription and mRNA injection and confocal imaging Histone2B-eGFP mRNA was transcribed with the mMES- SAGE mMACHINE SP6-Kit (Ambion). The template for in-vitro transcription was obtained via PCR from the pCS2P-plasmids backbone in which the Histone2B-eGFP sequence was cloned. Primers 59-ATTTAGGTGACACTATAG-39 and 39-CAGGAA- ACAGCTATGACCATGATTACG-59 were used for template amplification. Medaka eggs were injected at the late stage 0. For imaging, embryos were mounted in 0.7% low melting point agarose in H2O on a coverslip that was glued into the hole of a microscope slide. The eggs were orientated with the animal pole facing down and subsequently scanned with a Nikon C1 confocal microscope with a 4.107 minute delay between each stack. Nuclear size was determined as the area measured by the contrast based ‘‘Find 2D Nuclei’’ measurement protocol in the Volocity software on maximum intensity projections of confocal stacks. Expression analysis and RT-PCR Total RNA of medaka embryos at different stages was isolated as previously described [31]. RNA amounts for reverse transcrip- Asynchronous Cell Division in Medaka before MBT tion were measured with the Qubit-Kit (Invitrogen). Investigated stages were stage 0–2 (1–4 cells), stage 8–10 (64–1000 cells), stage 11 and stage 24. Quantitative real-time PCR was performed with SYBR Green reagents in a reaplex2 Mastercycler (Eppendorf). Target genes were orthologs of zebrafish genes for which up- regulation in zebrafish embryos after the 64-cell stage had been reported [7]. Used primer pairs were intron-spanning and are listed in Table S2. All results are averages of three independent experiments. Error bars represent standard deviation of the mean. Relative expression levels were calculated after correction to ef1a1 expression, which was used as housekeeping gene. Statistical analysis Significance values for cell division desynchronization and upcoming metasynchronous cell division, for asymmetry compen- sation, for symmetry/metasynchrony correlation and for asym- metry/cell-volume correlation were calculated using the Chi- square test. Significance values for RT-PCR were calculated with the Student’ t-test. Significance values for polymerase II phosphorylation was calculated with the Welch’ t-test. A probability of p,0,05 was considered as statistically significant; * and ** and *** represent statistical significances below p,0.05, p,0.01 and p,0.001 respectively. Supporting Information Figure S1 Hoechst staining at cell division from 128 to 256 cells. (A–C) Different time points (early-late) during cell division from 128 cells to 256 cells. (A) Interphase. All cells are in interphase (yellow arrows). (B) Early phase. Cell division starts first in central cells (green arrows), while peripheral cells do not yet divide (yellow arrows). (C) Late phase. Central cells have finished mitosis (yellow arrows) when peripheral cells undergo mitosis (green arrows). (TIF) Figure S2 Cell divisions at mid-late cleavage phases of a symmetric dividing embryo after H2B-eGFP-injection. Cell division progression of an embryo that has divided symmetrically from 2 to 4 cells at 4 successive cell cycles. (A) Embryo at three different time points (interphase, early-, late- phase) at cycle 6, 32 to 64 cells. Early cell division appears in random positioned cells of the embryo at early phase (green arrows). Other cells have not entered interphase (yellow arrows). (B–D) Embryo at three different time points (interphase, early-, late- phase) at cycle 7 (64 to 128 cells), cycle 8 (128 to 256 cells) and cycle 9 (256–512 cells), respectively. Cell division starts first in central positioned cells (green arrows) during early phase of the embryos cycle. Peripheral cells have not yet entered cell division (yellow arrows). Central positioned cells have started to enter interphase (yellow arrows) and peripheral cells are still in ana-/ telophase of the cell cycle at later phase of the embryos cycle. (TIF) Figure S3 Nucleus size progression during cleavage phase. Progression of nuclear sizes (as an area measurement) in a mid-interphase cell at the 8 cell stage and in its derived daughter cells until to the late 64 cell stage. The nuclear area was determined every 4.107 min for each nucleus for 45 consecutive time points. Nuclear areas are large at mid-interphase cells, small before cell division and smallest after cell division. No detectable desynchronization until measurement point 27 (red circles). Early mitosis in cells at more central position (blue circle). Chromosomes are condensed in late cells, but not separated (red arrows). Nuclear 2 size is shown on the Y-axis (in mm ), measuring points at the X- PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e21741