(A) Nissl staining of 6 wk old adult Control and cDKO brain sections (P0-tamoxifen^ind). Note the decreased cDKO lateral ventricle (LV) size when compared to cDKO in 2A. Also note the abnormal stratified medial (MW) and lateral (LW) ventricular walls. (B) IHC staining of 6 wk old adult Control and cDKO LV MW and LW. Control MW ependymal cells are GFAP^- and S100β⁺, but some remodeled mutant MW highly expressed both GFAP and S100β. Both Control and cDKO LW SVZ showed MASH1⁺ transient amplifying C cells (arrows) and Doublecortin (DCX⁺) neuroblasts, but the mutant neurogenic niche is separated from the ventricular surface (dashed lines) via a much thicker wall. Nuclei labeled with DAPI (blue). (C) EM images of remodeled 6 wk LW. Instead of normal ependyma (E), the cDKO mutant mice showed abnormal free space (*) in the remodeled wall adjacent to the LV. (D) β-gal staining of 6 wk old rosa26r^{−/ −}, Control and rosa26r^+/−, cDKO mice injected with tamoxifen at P0. Control mice showed significant numbers of β-gal⁺ cells in the ependymal layer (E) and the SVZ, but the cDKO mice had very few β-gal⁺ cells along the remodeled walls. Scale bars: (A) brain section = 1 mm, close-up = 20 μm; (B) 20 μm; (C) 5 μm; (D) 50 μm.

(A) Nissl staining of nestin-creER^tm4, Numb^F/F, Numbl^+/- (Control) and nestin-creER^tm4; Numb^F/F, Numbl^−/− (cDKO) brain sections with P0-tamoxifen^ind. Sections of P7 and P14 brains showing lateral ventricle (LV) at or just rostral to the anterior commissure. Note the increased LV size in mutant mice (asterisks). MW: medial wall; LW: lateral wall. Quantitative analysis of LV size, represented as % of total LV area in section just rostral to AC, over total brain section area (to control for variations in brain section sizes during processing). *: P < 0.01, Wilcoxon Two Sample Test; n = 5 in each group; mean ± standard deviation (SD). (B) Close-up views of Nissl-stained LV MW ependymal cells from P7 and P14 control and cDKO sections after P0 induction. Arrows point to ependymal cells (E), showing gaps (arrowheads) in P7 and detachment (arrow) in P14 cDKO samples. CP: choroid plexus. (C) Close-up views of LV LW neurogenic niche. Arrowheads point to gaps at P7, and arrow points to denuded area at P14 in cDKO samples. (D) Antibody staining of P7 mutant LW neurogenic niche. S100β⁺ ependymal cells were made postnatally in mutant mice. Nuclei labeled with DAPI (blue). Scale bars: (A) P7, P14 = 1 mm; (B, C, D) 20 μm.

(A) Nissl and antibody staining of P14 cDKO lateral ventricle (LV) lateral wall (LW). Nissl staining showing cellular aggregate on damaged LW (arrowhead). These aggregates stain positive for Numb and GLAST, representing cells that escaped CreER-mediated recombination (arrowheads). They also express GFAP, and are highly proliferative as shown by transient 1 hr BrdU pulse (arrows). (B) EM images of P14 Control and cDKO LW lining. Unlike Control LW, lined with ciliated ependyma (E, arrowheads), mutant LW showed clumps of transient amplifying C cells bordering LV lumen (C, arrows). (C) IHC staining of Control and cDKO mice injected with tamoxifen (T) at P0, pulsed with BrdU (B) at P16, then sacrificed (S) at 6 wks of age. In Control LW, the ependymal wall (dash line) is largely GFAP^- and GLAST^-, and the SVZ showed very sparse BrdU incorporation throughout. In the remodeled cDKO LW, there are many BrdU⁺ cells that are also GFAP⁺ or GLAST⁺ (arrows). Quantitative analysis of LW BrdU⁺ cells: from single coronal section at or just rostral to anterior commissure. (D) Nissl stain of 6 wk brain sections from Control and cDKO mice injected repeatedly with tamoxifen. Note the ventricular enlargement (asterisk) in cDKO mice, due to poor ventricular wall structural integrity (arrow). Quantitative analysis of 6 wk old LV size, represented as % of total LV area in coronal section just rostral to anterior commissure, over total brain section area. For all quantitative analysis: *: P < 0.01, Wilcoxon Two Sample Test; n = 5 mice in each group; mean ± SD. Scale bars: (A, C) 20 μm; (B) 10 μm; (D) brain section = 1 mm, closeup = 20 μm.

(A) EM images of control and cDKO mutant ependymal cells (P0-tamoxifen^ind). Toluene blue staining of P7 semi-thin sections showing the ependymal layer (E, arrows) and apical cilia (arrowheads). EM images of P14 ependymal cell borders showing interdigitation in control cells (yellow arrowheads), but a lack of interdigitation (yellow arrows) and cell separation (asterisk) between mutant ependymal cells. (B) Antibody staining of P7 Control and cDKO mutant lateral ventricle (LV) medial wall (MW) ependymal cells (P0-tamoxifen^ind). Note the lack of apical Phalloidin staining in mutant cells (arrowheads), low level of border E-cadherin staining in mutant cells except for an occasional Numb-expressing cell (arrows), and co-localization of E-cadherin and Numb staining in control cells (arrowheads). (C) Antibody staining of control P0 and P14 MW ependymal cells, showing upregulation of cell-border E-cadherin during maturation. (D) Co-immunoprecipitation (IP) of E-cadherin and FLAG-Numb. Protein extract from postnatal ependymal cells electroporated with FLAG-Numb expression construct was subjected to IP with beads and antibodies as indicated. Grin1 was used as positive control for Numb binding and negative control for non-specific E-cadherin interaction. The upper and lower panels were immunoblotted (IB) with anti-FLAG and anti-E-cadherin (E-cad.) antibodies, respectively. Note that E-cadherin was part of the FLAG-Numb IP complex, and vise versa. (E) Nissl, Doublecortin (DCX), and TUNEL staining of P7 Control and cDKO brain sections (P0-tamoxifen^ind). Note the greatly increased striato-cortical junction (SCJ) cellularity in the cDKO sample. DCX and TUNEL co-staining on P7 Control and cDKO brain sections shows greatly increased numbers of apoptotic neuroblasts in the mutant SCJ. (F) Nissl, DCX, and TUNEL staining of P14 CALSL-NICD; nestin-creER^tm4 mice either with (+) or without (-) P0-tamoxifen^ind. Note the greatly increased SCJ cellularity, as well as apoptotic DCX⁺ neuroblasts after tamoxifen^ind. Nuclei labeled with DAPI (blue). Analysis of SCJ area and TUNEL⁺ cells: SCJ from single coronal section just rostral to anterior commissure; n = 5 mice in each group. For all quantitative analyses: *: P < 0.01, Wilcoxon Two Sample Test; mean ± SD. Scale bars: (A) Semi-thin = 10 μm; P14 = 500 nm; (B, C) 20 μm; (E, F) SCJ Nissl = 100 μm, brain sections = 1 mm, DCX/TUNEL = 50 μm.

(A) Diagram of the Nestin-CreER^tm construct. (B) β-gal staining of nestin-creER^tm; r26r embryos: E12.5 embryos with or without E10.5 tamoxifen induction (tamoxifen^ind). Line 4 and 8 are 2 independent lines (Cortex, 30 μm cryostat section). Line 4 without tamoxifen showed no detectable recombination. (C, D) β-gal staining of nestin-creER^tm4; r26r brain sections. (C) 5 week (wk) old nestin-creER^tm4; r26r mice with or without single tamoxifen^ind at P0 or P7 (50 μm vibratone section). Arrows in coronal sections point to lateral ventricles (LV). Enlarged views of LV (Lat. Vent.) showing both medial (MW) and lateral wall (LW). Note the lack of β-gal⁺ cells in choroid plexus (CP). (D) Single dose P14-tamoxifen^ind in nestin-creER^tm4; r26r mice assayed at 1 wk or 1 month post-injection. Note the increase in β-gal⁺ cells in olfactory bulb (OB) and rostral migratory stream (RMS, arrows) in 1 month post-injection sample (50 μm vibratone sagital section). (E) Co-labeling of β-gal⁺ and BrdU⁺ SVZ cells. nestin-creER^tm4; r26r mice were tamoxifen induced at P14. 2 wks later, proliferating SVZ cells were labeled with 3-day BrdU pulse (arrows). 50 μm floating vibratone sections were stained for β-gal activity followed by BrdU immunohistochemistry. β-gal staining was inverted to dark-field red channel to create the merged image. (F) Electron micrographs of β-gal⁺ SVZ neuroprogenitor and ependymal cell from nestin-creER^tm4; r26r mice injected with tamoxifen at P14 and analyzed 1 wk later. Note the electron dense β-gal enzyme granules surrounding the nuclei (red asterisks), as well as the deeply invaginated nucleus of SVZ astrocytic progenitor (red arrow) and the apical cilia of ependymal cell (red arrowheads). Scale bars: (B) embryo = 1 mm, C. Cortex = 100 μm; (C) Coronal Sec. = 1 mm, Lat. Vent./O.B. = 100 μm; (D) 500 μm; (E) 50 μm; (F) 2 μm.