Asynchronous Cell Division in Medaka before MBT Figure 6. Quantitative gene expression analysis of Ccnb1, Rsp12 and PSMC1 during early embryogenesis. Expression levels of 3 candidate genes during 4 different time periods (Stages 0–2, Stages 8–10, Stage 11, Stage 14). (A) Expression of Ccnb1 is strong during stages 8–10 (p,0,0268) and stage 11 (p,0,0452). (B) Expression of Rsp12 shows induction at stage 11 and robust upregulation at stage 14 (p,0,0042). (C) Expression of PSMC1 is not upregulated. Expression levels at stages 0–2 were set as 1. (Significance was tested with the Student’s t-test, error bars are standard deviations). doi:10.1371/journal.pone.0021741.g006 next day, embryos were transferred to 30 ml of fresh PBS on a microscope slide, covered with a cover slip and scanned immediately with a Nikon C1 confocal microscope. For cell volume measurements, embryos were stained with the membrane dye CellMask DeepRed (Invitrogen (Available: CellMask DeepRed: C10046) http://products.invitrogen.com/ivgn/product/ C10046. Accessed 20 Jun 2011) in a 1:1000 dilution in PBS for 100 and washed in o/n in PBS at 4uC. Right before washing, DNA was stained by adding Hoechst in a 1:2000 dilution for 20. After washing, embryos were transferred into DABCO over night and were fixed in Mowiol on a microscope slide on the next day. Confocal images were taken using a Leica TCS SP5 confocal microscope. For p-polymerase II detection, embryos were fixed in PFA, stored in PBS at 4uC, removed from the yolk as described above. Embryos were permeabilised with 0.1% TritonX in PBS for 100, blocked in 5% BSA in PBS for 1–2 h and incubated with anti p- RNAPII antibody (SantaCruz (p-Pol II (8A7): sc-13583); Avail- able: http://www.scbt.com/datasheet-13583-p-pol-ii-8a7-antibody. html. Accessed 20 Jun 2011) at 1:1000 for 48 h at 4uC. After first antibody incubation, a 100 washing in PBS for 4 times at RT followed. Detection was done with an Alexa568-coupled anti mouse antibody at 1:1000 in 5%BSA for 24 h at 4uC. DNA was stained with Hoechst. Confocal imaging was done with a Nikon C1 confocal microscope. Cell cycle analysis Cell cycle synchrony was investigated in previously DNA stained and confocal imaged embryos. For visualization, confocal stacks were loaded into the Volocity V5.3.2 imaging software (PerkinElmer) and filtered. Embryos were classified as asynchronous if cells at different stages of the cell cycle (Interphase/Mitotic phase) were found within the embryo. Cells were considered as being in interphase as long as only 1 nuclear signal within the same cell was detected. PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e21741