Mitotic WNT signalling orchestrates neurogenesis in the developing neocortex (2021)
Figure 2

Figure 2. DKO embryos show delayed cell cycle progression and increased mitosis length in BPs.

(A) Triple-IF for BrdU, Pax6 and Tbr2 (not shown) in E12.5 control and DKO neocortices. BrdU was injected into pregnant dams one hour before sacrifice.

(B) Quantification of the proportion of Pax6+Tbr2- cells that are BrdU+, in control and DKO neocortices at E11.5, E12.5 and E13.5. Data are expressed as fold change vs. controls, with the control values set to 1.0, and are means ± SEM (E11.5 n=4 embryos, 3 litters; E12.5 n=5 embryos, 5 litters and E13.5 n=5 embryos, 4 litters).

(C) Average percentage of APs (Pax6+Tbr2-) (from B) that are BrdU+, in E13.5 control and DKO neocortices. Data are means ± SEM.

(D) Co-IF for Tbr2 and BrdU (double positive cells depicted by white arrowheads) in E12.5 control and DKO neocortices. BrdU was injected into pregnant dams one hour before sacrifice.

(E) Quantification of the proportion of Tbr2+ cells that are BrdU+, in control and DKO neocortices at E11.5, E12.5 and E13.5. Data are expressed as fold change vs controls, with the control values set to 1.0, and are means ± SEM (E11.5 n=4 embryos, 3 litters; E12.5 n=5 embryos, 5 litters and E13.5 n=5 embryos, 4 litters).

(F) Average percentage of BPs (Tbr2+) (from E) that are BrdU+, in E13.5 control and DKO neocortices. Data are means ± SEM.

(G) Double IdU/BrdU injection strategy used to calculate S-phase length. Top, scheme. Bottom, triple IF for IdU/BrdU/Tbr2 to detect APs (Tbr2-) and BPs (Tbr2+) that have exited S-phase (IdU+/BrdU-, white arrowheads) in control and DKO E13.5 neocortices.

(H) Average percentages (numbers in bar graphs) of the IdU+ cells that are either BrdU+ or BrdU- , scored as in (G), for control and DKO APs and BPs. Data are means ± SEM (n=4 embryos, 3 litters).

(I) S-phase length of E13.5 control and DKO APs and BPs, calculated from the data of panel (H, yellow) as described in Appendix Methods. Data are means ± SEM (n=4 embryos, 3 litters).

(J) Total cell cycle length of E13.5 control and DKO APs and BPs, calculated from the data of panels (C), (F) and (I) as described in Appendix Methods. Data are means ± SEM (n=4 embryos, 3 litters).

(K) Co-IF for the proliferation marker Ki67, the mitotic marker phospho-histone H3 (pHH3), and Tbr2 to identify mitotic APs (division at apical surface) and BPs (division in basal VZ or SVZ) in control and DKO E13.5 neocortices. Arrowheads depict pHH3+Tbr2+ cells.

(L) Average percentage of Ki67+ APs and BPs that are pHH3+ in control and DKO E13.5 neocortices. Data are means ± SEM (n=4 embryos, 3 litters).

(M) Mitosis length of E13.5 control and DKO APs and BPs, calculated from the data of panels (J) and (L) as described in Appendix Methods. Data are means ± SEM (n=4 embryos, 3 litters).

(N) IF for cleaved caspase 3 (Casp3) to identify apoptotic cells in E13.5 control and DKO neocortices.

(O) Quantification of apoptotic cells in control and DKO neocortices at E11.5, E12.5 and E13.5. Data are represented as number of Casp3+ cells per 5000 cells. Data are means ± SEM (E11.5 n=4 embryos, 3 litters; E12.5 n=3 embryos, 3 litters, and E13.5 n=4 embryos, 3 litters).

Data information: All scale bars 20 μm. Unpaired two-tailed t-test for all statistical analyses: ns, not significant; * p < 0.05, ** p < 0.01. p = 0.77 (B, E11.5); p = 0.16 (B, E12.5); p = 0.030 (B, E13.5); p = 0.014 (C); p = 0.017 (E, E11.5); p = 0.010 (E, E12.5); p = 0.013 (E, E13.5); p = 0.089 (F); p = 0.0069 (H, APs); p = 0.0078 (H, BPs); p = 0.018 (I, AP); p = 0.027 (I, BP); p = 0.43 (J, AP); p = 0.016 (J, BP); p = 0.35 (M, AP); p = 0.012 (M, BP); p = 0.24 (O, E11.5); p = 0.56 (O, E12.5); p = 0.26 (O, E13.5).