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. 2020 Oct 5;21(11):e50944. doi: 10.15252/embr.202050944

Figure 4. Mitosis is more frequent at the PS, and PS mitotic rounding is not always apical.

Figure 4

  • A
    Graphic representation of a transverse section from an E6.5 embryo. The epiblast is divided into three distinct regions: anterior (red), posterior (green) and PS (blue). The PS region is defined as the area where the basement membrane (yellow) is degraded.
  • B
    Representation of cell and mitosis disposition at the PS. Apical mitoses are in red, non‐apical mitoses in cyan and mesenchymal mitoses in green. Mitosis is considered non‐apical when occurring away from the epiblast apical border at a minimum distance corresponding to the size of one nucleus (± 10 μm), in a cell that has not crossed the perforated basement membrane, retains its apical attachment and does not display a mesenchymal phenotype.
  • C
    Z‐projections of transverse sections from E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. Samples were stained for F‐actin (Phalloidin, grey) and mitosis (Phh3, magenta) on the left and nuclei (DAPI, cyan) and basement membrane (collagen IV, yellow) on the right. Yellow arrowheads show non‐apical mitosis. White arrows delimitate borders of the basement membrane degradation region. Scale bars: 25 μm. A, anterior; P, posterior.
  • D
    Mitotic Index (Phh3/DAPI) in percentage at the anterior, posterior and PS regions of E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. The posterior region quantification excludes counts from the PS region. Normality was assessed using a Shapiro–Wilk test followed by either two‐tailed t‐test or Mann–Whitney test. ns, non‐significant, *P‐value ≤ 0.05, **P‐value ≤ 0.01 and ***P‐value ≤ 0.001.
  • E
    Ratio of non‐apical mitosis (non‐apical Phh3/Phh3, normalized by number of nuclei) in percentage at the anterior, posterior and PS regions of E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. Normality was assessed using a Shapiro–Wilk test followed by Mann–Whitney tests. ns, non‐significant, *P‐value ≤ 0.05, **P‐value ≤ 0.01 and ***P‐value ≤ 0.001. Data information (D, E): values are shown as Mean ± SEM. E6.5: n = 18 sections from six embryos, E6.75: n = 11 sections from five embryos, E7: n = 18 sections from four embryos. E6.5 n cells: anterior = 730, posterior = 445, PS = 221. E6.75 n cells: anterior = 518, posterior = 259, PS = 115. E7 n cells: anterior = 1,324, posterior = 712, PS = 296.
  • F
    Violin plots representing the distribution of cells in G2/M-phase in cells with high, compared with low, Brachyury expression, among cells annotated as primitive streak and mesoderm harvested from E6.5, E7 and E7.25 embryos. Violin plots are overlaid with white box plots marking 25th percentile, median and 75th percentile. Data information (from Pijuan‐Sala et al, 2019): E6.5: three biological and two technical replicates; E7.0: six biological and three technical replicates; E7.25: three biological replicates. *P‐value ≤ 0.05, **P‐value ≤ 0.01 and ***P‐value ≤ 0.001 using Student's t‐test.

Source data are available online for this figure.