Fig. 6
Relationship of the transcription compartment with the nuclear architecture. a Confocal Airyscan image of 512-cell stage embryos co-injected with miR430 MOs (green) and either mRuby3:H2B (top) or mApple:PCNA (bottom) fusion proteins (red). Arrows indicate low density chromatin at the site of miR430 activity. b Time-lapse of 512-cell stage nucleus, injected with mApple:PCNA fusion proteins (red). Image shows individual z-slices from within the nucleus exhibiting two PCNA deficient regions. Graph below shows the evolution of the individual depicted PCNA deficient regions over time (A & B), in addition to deficient regions from other 512-cell stage nuclei from the same embryo, detected using spot detection plugin in Icy Bioimage. Time indicates duration from first detection of PCNA signal within the cell cycle in seconds and minutes. c Charts representing the transcription focus volume over time based on miR430 MO signal, measured using spot detection plugin Icy Bioimage software, top panel: 128-cell stage, bottom: 512-cell stage. Boxplots on the right show variation in peak volume. Middle line = median. whiskers = nearest hinge ± 1.5 * interquartile range. d Nascent RNA detection (green) by ethynyl-uridine incorporation and subsequent Alexa fluor 488 tagging of embryos fixed in 4 min intervals within 512-cells stage, demonstrating different stages of the transcription compartment structure during the cell cycle. e DNA content of 12-somite stage embryos untreated and heat-shocked was determined by propidium iodide DNA staining analysis. FACS traces show a shift in the position of the Gap phase 1 (G1) and Gap phase 2 (G2) peaks (marked), between the heat-shock untreated and treated groups, in accordance with a doubling in DNA content in the heat-shocked group. f Representative images of miR430 MO-injected diploid and induced polypoid 256-cell stage embryos (green). g Bar chart showing comparison of miR430 foci number between diploid and tetraploid embryos. n.RNA nascent RNA, S S-phase, P prophase, PM prometaphase; Scale bar, 5 µm. Source data for (b, c, g) are provided as a Source Data file.
