Asynchronous Cell Division in Medaka before MBT Figure 5. Polymerase II phosphorylation in embryos before MBT. Phosphorylation of mRNA polymerase II in medaka embryos at 8 to 16 cells (A), 32 cells (B), 64 cells (C) and 128 cells (D). (A) p-Pol II staining, first detected in cells during the late stages of mitosis between the 8-cell stage and the 16-cell stage. (B) p-Pol II signal in isolated cells at 16-cell stage. (C) 64-cell stage showing high levels of p-Pol II signal. Almost no phosphorylation is seen in peripheral cells, but ubiquitous signal in central cells. (D) 128-cell stage. Almost no p-Pol II positive nuclei were found in peripheral cells, most central nuclei stain positively. doi:10.1371/journal.pone.0021741.g005 Fishes Medaka fishes (Oryzias latipes) of the Carbio strain (Carolina Biological Supplies, USA) were kept as a large random-mating colony under standard conditions at a 14 h light-cycle and 24uC room temperature. Embryos were collected 20minutes after fertilization, extricated from the filament and raised in de-ionized water. Embryos where staged according to Iwamatsu [20]. Light microscopy Medaka embryos at the 4 cell stage were transferred into 2.5% methylcellulose and imaged using a M205FA Leica Microscope. Images were processed with ImageJ. Fluorescent staining and confocal microscopy Embryos were cooled down to 4uC to slow down the cell cycle and then fixed in 4% PFA/16PBS over night (o/n) at room temperature (RT). After fixation, embryos were rinsed in 16PBS for 200 at RT and stored in PBS at 4uC for 48 h. The chorion was physically removed using forceps and the embryos were detached from the yolk. For cell cycle measurements, DNA was stained with Hoechst (Invitrogen (Hoechst 34580: H21486); Available: http://products. invitrogen.com/ivgn/product/H21486?ICID = search-product. Ac- cessed 20 Jun 2011.) in a 1:2000 dilution in PBS for 20 and washed 2 times in PBS for 100 and additionally in fresh PBS o/n at 4uC. The PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e21741