The m5C reader Ybx1 regulates embryonic cortical neurogenesis by promoting progenitor cell cycle progression
Fig 4
Ybx1 regulates proliferation and differentiation of cortical progenitor cells.
(A) Coronal brain sections at P0 were stained with antibodies recognizing BrdU and Ki67. Pregnant mothers received a BrdU pulse 24 h before pup dissection at P0. Representative cortical regions were shown. Scale bar: 100 μm. (B and C) Quantification of BrdU+ cell numbers (B) and the percentage of cells exiting the cell cycle (C) at P0 shown in (A). All statistical data are presented as box and whisker plots. For BrdU+ cell numbers, Ctrl (n = 27 confocal fields) vs. cKO (n = 24 confocal fields), ***p = 2.11E−04; Het (n = 23 confocal fields) vs. cKO, ***p = 4.24E−04. For Ki67−BrdU+/BrdU+, Ctrl (n = 27 confocal fields) vs. cKO (n = 24 confocal fields), **p = 0.0015; Het (n = 23 confocal fields) vs. cKO, **p = 0.0051. (D) Coronal brain sections at E13.5 were stained with antibodies recognizing BrdU and Ki67. Pregnant mothers received a BrdU pulse 24 h before embryo collection. Representative cortical regions were shown. Scale bar, 50 μm. (E and F) Quantification of BrdU+ cell numbers (E) and the percentage of cells exiting the cell cycle (F) at E13.5 shown in (D). All statistical data are presented as box and whisker plots. For BrdU+ cell numbers, Ctrl (n = 22 confocal fields) vs. cKO (n = 25 confocal fields), **p = 0.0052; Het (n = 23 confocal fields) vs. cKO, *p = 0.012. For Ki67−BrdU+/BrdU+, Ctrl (n = 22 confocal fields) vs. cKO (n = 25 confocal fields), **p = 0.0028; Het (n = 23 confocal fields) vs. cKO, *p = 0.027. (G) Representative images of primary and secondary neurospheres formed by NSCs isolated at E13.5. Scale bar, 50 μm. (H–K) Quantification of the sizes (H, J) and numbers (I, K) of the primary (H, I) and secondary (J, K) neurospheres show in (G). All statistical data are presented as box and whisker plots. In H, Ctrl (n = 42 neurospheres) vs. cKO (n = 38 neurospheres), ****p = 6.65E−05; Het (n = 46 neurospheres) vs. cKO, ****p = 5.22E−05. In J, Ctrl (n = 42 neurospheres) vs. cKO (n = 38 neurospheres), ****p = 2.76E−05; Het (n = 46 neurospheres) vs. cKO, ****p = 2.26E−05. (L) Schematic drawings of the IUE experiment. Pregnant mice were injected at E14.5 with plasmids. Subsequently, a single EdU pulse was administrated at E15.5, and embryos were dissected at E16.5 for analysis. (M) Confirmation of Ybx1 knockdown in the cortex after IUE of shYbx1. Immunostaining for GFP and Ybx1 on coronal sections of E16.5 cortex. Representative cortical regions were shown. Scale bar, 50 μm. (N) Immunostaining for GFP, EdU, and Ki67 on coronal sections of E16.5 mouse cortex after knockdown of Ybx1 using IUE. Representative cortical regions were shown. Scale bar, 50 μm. (O and P) Quantification of percentage of EdU+GFP+/GFP+ (O) and Ki67-EdU+/EdU+GFP+ (P) shown in (N). Data are presented as box and whisker plots: in O, shCtrl (n = 22 confocal fields) vs. shYbx1 (n = 19 confocal fields), ***p = 1.38E−04; in P, shCtrl (n = 22 confocal fields) vs. shYbx1 (n = 19 confocal fields), ***p = 8.30E−04. At least 3 pups or embryos were analyzed for each genotype or condition. Analyses were performed by one-way ANOVA followed by Tukey’s multiple comparison test (B, C; E, F; H–K), or by unpaired Student t test (O, P). ns, not significant. The data underlying all the graphs shown in the figure are included in S1 Data.