Figure 6

Barr body formation at onset of XCI in early differentiating female XX ESCs. (A) Temporal evolvement of the Barr body in relation to Xist RNA spreading and RNAP II exclusion. Undifferentiated: Xist/Tsix RNA transcripts from both X chromosomes before onset of XCI. The fibrillar appearance of RNA signals point to a high local concentration of transcripts around a strong RNAP II signal at each of the two Xist (Tsix) loci. Note that RNAP II and RNA expand into the ANC between the denser packed CDCs. Scale bar: 2 μm, insets 0.5 μm. Day 3: focal spreading of Xist RNA into a confined nuclear region without visible chromatin compaction containing abundant RNAP II signals throughout the loose ‘Xist RNA territory’. Scale bars: 2 μm, insets 1 μm. Day 4: RNAP II signals mostly at the edge of the ‘Xist RNA territory’. Chromatin shows first signs of compaction. Scale bars: 2 μm, insets 1 μm. Day 5 and day 9: Clear outline of a Barr body with distinct chromatin compaction underneath the Xist RNA territory and exclusion of RNAP II. Scale bars: 2 μm, insets 0.5 μm. (B) Histograms of DAPI intensity differences (plotted as over/underrepresentation for each class) in the emerging Barr body as compared to the entire nucleus. Mean differences from at least ten nuclei from each state are indicated with standard deviations documenting the shift towards higher intensity classes at day 5 upon differentiation and an additional shift to the right at day 9. (C) Boxplots showing the number and volume distributions of discernible Tsix/Xist RNA foci (quantified by Volocity) from single Xi territories in undifferentiated XX ESCs. Day 0, n = 13; day 5, n = 14; and day 9, n = 12. Data for C2C12 (compare Figure 4) are shown for comparison. ANC, active nuclear compartment; CDC, chromatin domain cluster; DAPI, 4',6-diamidino-2-phenylindole; ESC, embryonic stem cell; RNAP II, RNA polymerase II; XCI, X chromosome inactivation; Xi, inactive X chromosome; Xist, X inactive specific transcript.