(A) Schematic representation of Notch genomic deletion in the migrating neurons. (B-E) Venus immunostaining 6 days post-electroporation with pTα1-Cre-IRES-Venus (B-D) or pTα1-IRES-Venus (E) into indicated mice. Bar (μm) = 100. Dashed lines indicate pial and ventricular surfaces. (F) The graphs indicate quantification of the distribution of Venus⁺ neurons in the 10 bins dividing the whole thickness of the cortex as indicated in B in each genotype. The data represent the mean ± SEM of 4 brains each from independent experiments. p<0.0001, p<0.0001, and p>0.05 for rl/rl, Notch1^fl/fl;Notch2^fl/fl(+Cre) and Notch1^fl/fl;Notch2^fl/fl(−Cre), respectively compared with wild-type by Two-sample Kolmogorov-Smirnov test (K-S test). F(9,54)=107.40, p<0.0001, F(9,45)=54.92, p<0.0001, and F(9,54)=0.78, p>0.05, respectively by Repeated Measures ANOVA.

(A-C) Venus immunostaining revealed migrating neuronal morphology 3 days post-electroporation with pTα1-Cre-IRES-Venus in wild-type, Reeler and Notch1 ^fl/fl; Notch2 ^fl/fl mice. Red arrows in (C) indicate ectopic primary processes. (A’-C’) 3D reconstruction of Venus⁺ migrating neurons in mice of each genotype 3 days post-electroporation. (D) Percentage of primary processes (directly protruded from cells) oriented normally (The ‘oriented normally’ primary processes were defined according to their angle towards the pial surface within ±15°) and abnormally. * p<0.01, **p<0.001, Student’s t-test comparing with wt. (E) Percentage of cells with one (black), two (blue) or >3 (white) processes per cell. * p<0.05, **p<0.01, Student’s t-test comparing corresponding bins to wt. (F) Box plots of the average primary process length per cell (total process length/number of the primary processes). *p<0.05, **p<0.01, Mann-Whitney’s U test. A total of 120 cells/genotype from four brains (different litters) were analyzed for orientation (D) and number of processes (E). The box plots of primary process length (F) were obtained for each genotype (n=30 each from 3 brains {different litters}). Bars (μm) = 10.

(A) Venus⁺ neurons 3 days post-electroporation with pTα1-Cre-IRES-Venus in rl/rl;LSL-Notch ICD mice. Left neuron in (A) showed rescued morphology with processes that were more pial-oriented compared to rl/rl (e.g. Fig. 4B). (A’) 3D reconstruction of Venus⁺ neurons (compare with rl/rl in Fig.4B’). (B-D) Quantification of direction of primary processes (B), primary process number per cell (C) and average primary process length per cell (D) for the electroporated neurons in mice of indicated genotypes. *p<0.05, Student’s t-test (B, C), *p<0.05, Mann-Whitney’s U test (D). A total of 120 cells/genotype from four independent experiments were analyzed for orientation (B) and number of processes (C). The box plots of primary process length (D) were obtained for each genotype (n=30 each from 3 independent experimental sets). For better comparison with Reeler, duplicated data from Figure 4 were included. Bar (μm) = 10.

(A) Effects of active Dab1 on Notch ICD activity. Relative Rbpj luciferase activity of each condition is compared to lane 1 set as 1.0. The indicated values are the mean ± SEM of three experiments performed in triplicate. *p<0.05, Student’s t test. (B) Cos-7 cells were transfected with indicated plasmids, and the expression of Notch1 ICD and GFP (as an internal control, reprobed on the same membrane) were analyzed by IB. The graphs show the relative intensity of the Notch1 ICD against the left lane after normalization with GFP as a transfection control. The data represents the mean ± SEM of 3 independent experiments. *p<0.01 paired t-test. Transfection of caSrc without Dab1 or Dab1 without caSrc constructs gave equivalent results to Dab1 5YF with/without caSrc. Transfection of caSrc with/without Dab1 constructs did not alter both endogenous and exogenous Fbxw7 expression levels (data not shown). (C) IB of lysates from rl/rl and rl/+ brain slices against polyubiquitin (FK2) and Notch1 after immunoprecipitation with a Notch1 antibody. IB against GAPDH was obtained from the flow-through of IP. The samples in the left 3 lanes were obtained from freshly prepared cortical slices, and the samples in right 3 lanes were from slices cultured with proteasome inhibitors. Note the decrease of precipitated non-polyubiquitinated Notch1 in Reeler (lanes 1’ and 4’), consistent with the increase of polyubiquitinated Notch1 (lanes 1 and 4). Arrow indicates p100-120 Notch1 ICD bands. Asterisk shows IgG bands. (D) IB of lysates from cultured cortical neurons transfected with the indicated plasmids against polyubiquitin (FK2) and myc after IP with a myc antibody. The transfected neurons were exposed to Reelin containing- or mock medium for 6 hours with protease inhibitors before collection. IB against GAPDH was obtained from the IP flow-through.

(A-H) Nuclei (red) and Notch1 (green in A-D’, antigen is Notch1 intracellular domain) and Notch1 ICD cleaved form (green in E-H) immunostaining in the cortex of wild-type and mutants as indicated. Higher magnifications are shown in the insets. Dashed lines in (E-G) indicate the pial surface and the border between CP and MZ in wild-type or superplate (SPP) in the mutants. (G) and (H) show upper CP and lower CP/IZ, respectively (the asterisk indicate the same cell). Note that Notch1 ICD expression at the lower CP (inverted layer II/III) is significantly decreased in Vldlr/ApoER2 mutant (H) which is similar to Reeler (not shown). Green staining at the pial surface in (G) is nonspecific. Bars = 25 μm. (I) Immunoblots of full-length Notch1 (p300), Notch1 ICD and β-actin from the same cortical lysates of 2 rl/+ and 2 rl/rl. Notch1 ICD bands include p110 non-phosphorylated and p120 phosphorylated forms. Right panel: Relative values of the band intensity of indicated proteins from rl/rl against those from rl/+, which were set as 100. Band intensities for each mutant were normalized to β-actin. The data represent the mean ± SD of 5 brains from independent experiments. *p<0.01 by paired t-test. (J) Immunoprecipitation (IP) of brain lysates with a non-specific goat IgG as negative control (left) or Dab1-specific antibody (E-19; right) followed by Notch1 and Dab1 immunoblotting (IB).

(A-D”) Venus (green) immunostaining with BrdU or Cutl1 (red or white) staining in indicated mutants at P14. Note that Cutl1 analysis was performed in the dorsal somatosensory region where endogenous Cutl1 expression in lower layers is normally absent. BrdU⁺/Venus⁺ or Cutl1⁺/Venus⁺ neurons in the Notch deleted cortex (C-D’, arrows) are abnormally positioned in lower layers than in the control cortex (A-B’, arrows). (C”, D”) Higher magnification views of BrdU⁺/Venus⁺ and Cutl1⁺/Venus⁺ double-labeled neurons around layer V, respectively (arrows). Bars (μm) = 100 (A-D”), 10 (C”, D”’). (E) Quantification of the distribution of Venus⁺ neurons under Layer IV in the Notch1 ^fl/+; Notch2 ^fl/+ and Notch1 ^fl/fl; Notch2 ^fl/fl cortex. The data represent the mean ± SEM of 4 brains each from independent experiments. * p<0.005, Student’s t-test.

(A-D) Immunostaining for Venus (black, green) and Tbr1 (red) in cortical slices of indicated genotypes 4 days (A-C’) or 4.5 days (D) post-electroporation (with pTα1-Cre-IRES-Venus). (D) Higher magnification view around the SPP. Dashed lines in (C’) and (D) indicate the pial surface and the border between the SPP and CP, respectively. Bracket in (C’) shows the SPP region. Note that electroporated Venus⁺ cells did not change their fate to Tbr1⁺ early-born neurons (C’, D). (E) Quantification of neuronal distribution showed significantly more cells in upper CP of rl/rl;LSL-Notch ICD compared to rl/rl cortex (K-S test, p<0.0001; Repeated Measures ANOVA, F(9,54)=18.91, p<0.0001). The data represent the mean ± SEM of 6 brains each. (F, F’) Immunostaining for Venus (green) and BrdU (red, white) in P3 cortical slices of rl/rl;LSL-Notch ICD electroporated with pTα1-Cre-IRES-Venus. Note that Venus⁺/BrdU⁺ cells (indicated by bracket) located over BrdU⁺ cells in surrounding lower layers. (G, G’) Venus immunostaining in P3 cortical slices of rl/rl and rl/rl;LSL-Notch ICD electroporated with pTα1-Cre-IRES-Venus. (G”) Quantification of Venus⁺ neurons located in the upper part of the CP (within the bin 5 indicated by a red bracket in G, G’. Entire thickness of the cortex was subdivided into 5 bins.) The data represent the mean ± SEM of 3 brains each. *p<0.05, Student’s t-test. Bars (μm) = 100 (A-C’, F-G’), 20 (D).

(A-C) Venus⁺ neurons were detected by immunohistochemistry 6 days after electroporation with pCDNA3.1 empty (A) or containing Dab1 5YF (B, C) plasmid with pTα1-IRES-Venus (A, B) or pTα1-Cre-IRES-Venus (C) plasmid in LSL-Notch ICD mice. Note that 5YF arrested many neurons beneath the CP which was mitigated by overexpression of Notch ICD. (D) Quantification of neuronal distribution in the LSL-Notch ICD cortex electroporated with indicated plasmids (K-S test, p<0.05; Repeated Measures ANOVA, F(9,126)=5.24, p<0.05). The data represent the mean ± SEM of 6 brains each. (E-G) Venus⁺ neurons were detected by immunohistochemistry at P14 after E14.5 electroporation with pCALSL empty (E), containing Notch ICD (F) or caRbpj (G) with pTα1 -Cre-IRES-Venus and pCDNA3.1-Dab1 5YF plasmids in wild-type mice. Note that compared to control (E), overexpression of Notch ICD (F) or caRbpj (G) resulted in fewer neurons located beneath layer IV. (H) Percentages of Venus⁺ neurons below layer IV in P14 wild-type cortex electroporated with indicated plasmids. The data represent the mean ± SEM of 10, 8, and 11 brains, respectively. *,**p<0.01, 0.05, by Student’s t-test. Bars = 100 μm.