Aspm knockdown decreases neural progenitor proliferation in the developing cortex. (A) Images of E15 mouse cortices electroporated at E12 with nontargeting (top panels, Control) or ASPM-directed small hairpin (bottom panels, shASPM) and GFP expression plasmids. Images were stained for GFP and either Hoechst (left) or Tuj1 (right). Dashed lines in the left panels represent the CP/IZ (top) and IZ/ SVZ (bottom) borders. Bar, 50 μm. (B) Distribution of cells across cortical zones 72 h post-electroporation at E15. (C) Fraction of GFP⁺ cells that overlap with Tuj1 staining in slices. (D) Images of E15 cortices electroporated with control small hairpin (left) or shASPM (right) plasmid. The left images in each set (see panels i,v) show the full span of the cortex. The right images in each set show GFP (panels ii,vi), PHH3 (panels iii,vii), or a merge of GFP and PHH3 (panels iv,viii) from the boxed area on the left. Asterisks indicate GFP, PHH3 double-positive cells. Bar, 25 μm. (E) Mitotic index of the GFP-positive cell population 72 h post-electroporation as measured by PHH3 staining. (F) Images of E15 cortices electroporated with control (left) or shASPM (right) plasmid. (Left panels) GFP staining. (Middle panels) Merge of BrdU (blue) and Ki67 (red). (Right panel) Merge of GFP with BrdU and Ki67. Arrowheads mark GFP, BrdU double-positive cells. Arrows mark GFP, BrdU, Ki67 triple-positive cells. Bar, 50 μm. (G) A 24-h BrdU labeling index as measured at E15. (H) Cell cycle exit index measured at E15.

Aspm knockdown perturbs neuronal migration at late stages of corticogenesis. (A) Images of E19 cortices electroporated at E15 with control (top panels) or shASPM (middle and bottom panels) and GFP expression plasmids. Images were stained for GFP (left) and either Hoechst (middle) or FoxP2 (right). Dashed lines (middle) represent the CP/IZ (top) and IZ/ SVZ (bottom) borders. Bar, 100 μm. (B) Distribution of electroporated cells across cortical zones at E19. (C) Distribution of electroporated cells within the VZ and SVZ at E19. Upward-facing error bars and black stars denote significant differences in fraction of SVZ cells. Downward-facing error bars and red stars denote significant differences in fraction of VZ cells. (D) Percent of electroporated cells that overlap with FoxP2 staining in slices at E19.

Expression of stabilized β-catenin rescues defects caused by Aspm knockdown. (A) Fold Wnt-mediated luciferase expression over control (Ctl, non-Wnt-containing medium) in P19 cells transfected with a combination of control (Ctl) or ASPM (shA) small hairpin expression constructs and vector (vector) or stabilized β-catenin (S37A mutant, βcat) expression constructs following exposure to Wnt-containing medium (Wnt). (B) Images of E16 cortices after electroporation at E13 with either control or shASPM, pCAGIG vector or pCAG-β-catenin, and GFP expression plasmids. (Left panels) GFP alone. (Right panels) GFP and Hoechst. Dashed lines in the right panels indicate the CP/IZ border (top) and IZ/SVZ border (bottom). Bar, 50 μm. (C) Distribution of electroporated cells across cortical zones at E16. (D) Fraction of GFP⁺ cells found in the upper half of the CP. (E) Images of E19 cortices showing the distribution of GFP⁺ cells following electroporation of control or shASPM and either pNeuroD vector or pNeuroD-β-catenin. (Left panels) GFP alone. (Right panels) GFP and Hoechst stain. Bar, 100 μm. (F) Distribution of electroporated cells across cortical zones at E19.