Progressive mode of the stochastic G1/S transition in the notochordal cells during embryonic body elongation.

<p>(A) Fucci (fluorescent ubiquitination-based cell cycle indicator) labels nuclei of G1 phase cells red (orange) and those of cells in the S/G2/M phase green. During the cell cycle of cells expressing Fucci, two chimeric proteins, mKO2-zCdt1 and mAG-zGeminin, reciprocally accumulate in the G1 and S/G2/M phases <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003957#pcbi.1003957-Sugiyama1" target="_blank">[48]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003957#pcbi.1003957-SakaueSawano1" target="_blank">[49]</a> (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003957#pcbi.1003957-Sugiyama1" target="_blank">[48]</a> for detailed molecular construction). (B) Cecyil (cell cycle illuminated: a zebrafish line producing zFucci) embryos at the 17 somite stage. A Z-stacking lateral view of optical sections. The anterior view is to the left, the posterior is to the right. (C) A mid-sagittal optical section of a fixed Cecyil embryo at 22 hpf counterstained with phalloidin Alexa Fluor 647. (D, E) Mid-sagittal optical section of a fixed Cecyil embryo at 18 hpf. A DIC (differential integrative phase contrast) image (D) and fluorescence image (E) are shown. The posterior-most green cell (PGC) and anterior-most red cell (ARC) in the upper and lower notochordal cells are indicated by green and red arrowheads, respectively. The posterior tip of the precursor pool of the notochord and the last formed somitic border are marked by sky blue and blue arrowheads, respectively. (F–K) Mid-sagittal optical sections of a developing Cecyil embryo. DIC images obtained at three different time points (F–H) and their corresponding fluorescence images are shown (I–K). The colored arrowheads indicate the same structures of cells as shown in D and E. The white arrow indicates a single cell whose cell cycle phase was transitioning from G1 (I) to S (J and K). Embryonic stages are indicated by somite stages (s.s.). (L) The temporal changes in the red and green fluorescence intensity of the cell indicated by the white arrow in G–H are plotted. The time course of the live imaging observations and embryonic stages is indicated in minutes and somite stages (s.s.), respectively. The vertical broken lines indicate the observation times corresponding to the images in g–i. The crossing time point of the red and green lines is indicated by the yellow point (115 min, in 18 somite stages). This time was selected as the time at which the G1/S transition occurred (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003957#s4" target="_blank">Methods</a>). White scale bars: 100 <i>µm</i>. Anterior to the left, posterior to the right. Dorsal to the top, ventral to the bottom. s.s, somite stage.