Radial Glial Neural Progenitors Regulate Nascent Brain Vascular Network Stabilization Via Inhibition of Wnt Signaling
(A–D) Effects of orc3 deletion on radial glial division. BrdU labeling (in red) revealed no obvious defects in ventricular zone division at E13.5, but substantial reductions at E15.5, especially at the ventricular surface (arrowheads in C and D). (E–H) Effects of orc3 deletion on radial glial density. RC2 staining (in green) revealed substantial reductions in radial glial density at E15.5 and near complete loss at P0. Boxed areas in (E–H) are shown in (E′–H′). Also notice more severe loss of radial glia in the mutant medial cortex (right side in H). (I–J″) Effects of orc3 deletion on radial glial fate. Although reduced in number, mutant radial glia express normal levels of Pax6 (in red) (I and J) at E16.5. PH3 (in green) staining also revealed a reduced number of mitotic cells at the ventricular surface (I′ and J′). (K–L) Quantification of Pax6+ cell density and expression level. Significant differences were observed in Pax6+ cell density (p = 0.0003, n = 4) but not Pax6 staining intensity (p = 0.32, n = 20). Note cells are less densely packed in mutants. (M–N) Effects of orc3 deletion on intermediate progenitors. Tbr2 staining (red) revealed severe reduction in intermediate progenitors in mutants at E16.5. (O–P″) Cell cycle exit analysis at E15.5. BrdU was administered at E14.5 followed by staining for BrdU (red in O, P, O″, and P″) and Ki67 (green in O′, P′, O″, and P″) at E15.5. (Q) Quantification of Tbr2+ cells in the subventricular zone at E16.5. Significant reductions were observed in mutants at E16.5 (p = 3×10−15, n = 9). (R) Cell cycle exit indices at E15.5 and E16.5. Significant increases were observed in mutants at E15.5 (p = 0.0001, n = 5), but not at E16.5 (p = 0.39, n = 5). Scale bar (in D): 200 µm for (A–D), 50 for µm for (E–H), and 60 µm for (I–J″) and (M–P″).