All mice in this study were treated according to protocols reviewed and approved by the institutional animal care and use committees of Northwestern University, Animal Study Protocol # 2010–1863, approved by the Northwestern University Office for the Protection of Research Subjects Institutional Animal Care and Use Committee. Timed-pregnant C57BL/6 mice were ordered from Charles River Laboratories (Wilmington, MA, USA). NcadFlox/Flox mice (B6.129S6(SJL)-Cdh2
/J) and Nes-Cre (B6.Cg-Tg(Nes-cre)1Kln/J) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). NcadFlox/Flox mice were genotyped using primers 5′-TGCTGGTAGCATTCCTATGG-3′ and 5′-TACAAGTTTGGGTGACAAGC-3′ as previously described . Nes-Cre mice were genotyped according to The Jackson Laboratory protocols available at http://jaxmice.jax.org/protocolsdb/f?p=116:2:903366808665603::NO:2:P2_MASTER_PROTOCOL_ID,P2_JRS_CODE:288,003771 (Cre). Axin2-d2EGFP reporter mice  (a gift from F Costantini, Columbia University Medical Center, New York, NY, USA) have been used to report β-catenin signaling in vivo. Axin2-d2EGFP reporter mice were genotyped as described .
Axin2-d2EGFP and Nes-Cre mice were mated to NcadFlox/Flox mice to generate (Axin2-d2EGFP; NcadFlox/+) and (NesCre; NcadFlox/+) F1 progenies, respectively. (Axin2-d2EGFP; NcadFlox/+) and (NesCre; NcadFlox/+) F1 progenies were mated to generate E12.0 embryos of (Axin2-d2EGFP; NesCre; NcadFlox/Flox) genotype. Littermate embryos of (Axin2-d2EGFP; NesCre; NcadFlox/+) and (Axin2-d2EGFP; NcadFlox/Flox) genotypes were used as controls without N-cadherin conditional knockout. Embryos were harvested at E12.0.
pSUPER.retro.puro driving shRNAs against N-cadherin (GACTGGATTTCCTGAAGAT; nucleotides 431–449 of mouse N-cadherin [GenBank:AB008811], which is identical to nucleotides 215–233 of human N-cadherin [GenBank:BC036470]) and control EGFP (CGATGCCACCTACGGCAAG; nucleotides 786–804 of GFP [GenBank:U55762]) were generously provided by M Wheelock (University of Nebraska, Lincoln, NE, USA). The shRNA to EGFP was used as the control for the Ncad-shRNA construct. pcDNA-Myr-AKT and DN-AKT (K197M) were kindly provided by Anna Kenney (Memorial Sloan Kettering cancer center 1275 York Ave., New York City, NY USA). The shRNA to eGFP was used as the control for the pSUPER Ncad-shRNA construct. Similar phenotypes were obtained with a second shRNA construct targeting mouse N-cadherin; mouse shRNAmir to N-cadherin in pSHAG-MAGIC 2c (pSM2c) retroviral vector was obtained from Open Biosystems (Thermo Scientific; 81 Wyman St. Waltham, MA 02454), accession number NM_007664, with oligonucleotide ID: V2MM_13176, target sequence: GCAGGCAAAGTTCCTGATATA. Control non-silencing shRNA (cat. RHS1703) is a negative control that expresses sequence TCTCGCTTGGGCGAGAGTAAG which has no homology to known mammalian genes. This non-silencing construct was used as a control to the two shRNAmir constructs.
In utero electroporation
For in utero injection, timed-pregnant mice at E13.5 were anesthetized using inhalation of isoflurane mixed in a constant ratio with oxygen, after which abdominal fur was removed, and the uterine horns were exposed through a midline laparotomy incision. DNA solution (2.5 µL) in H2O containing 0.0125% fast green was injected through the uterine wall into the lateral ventricle of the embryos using a glass micropipette made from a microcapillary tube. After injection, Tweezer-trodes (BTX, 84 October Hill Rd. Holliston, MA 01746–1388) were applied across the outside of the uterus, oriented to flank the embryonic brain, and five 50 ms square pulses of 39 V with 950 ms intervals were delivered by an electroporator (BTX 830). Following injection and electroporation, the uterus is returned inside the abdomen and the abdominal muscle wall and skin sealed with sutures.
To study the effect of N-cadherin knockdown on TOP-dGFP signaling in vivo, 0.8 µg/µL of DNA was used for pcDNA-Cre, pcDNA-lacZ, pCAG-mCherry and TOP-dGFP. To study the effect of DN-AKT on TOP-dGFP signaling in vivo, 0.25 µg/µl of pCAG-mCherry, 0.5 µg/µl of TOP-dGFP, and 1.0 µg/µl of DN-AKT or pcDNA3.0 were used. For migration and differentiation studies, 0.25 µg/µl of DNA for pCAG-GFP and 0.75 µg/µl of DN-AKT or pcDNA3.0 were used.
For the TOP-dGFP signaling studies, embryos were sacrificed 30 hours after electroporation. For the distribution and differentiation studies, embryos were sacrificed 24 hours after electroporation. Brains were fixed for 8 to 16 hours in 4% paraformaldehyde, cryoprotected in 30% sucrose dissolved in PBS overnight, and embedded in OCT (Optimal Cutting Temperature compound). A cryostat was used to make 12 µm coronal sections.
For studies of TOPdGFP expression in NcadFlox/Flox mice, histograms in Figure 1B were derived from three brains, 312 cells (mCherry, pcDNA-lacZ control), and three brains, 261 cells (mCherry, pcDNA-Cre). To determine the effect of DN-AKT on TOPdGFP in C57/B6 mice, three brains, 522 cells (mCherry, DN-AKT), and three brains, 645 cells (mCherry, pcDNA3.0 control), were analyzed. Cell distribution histograms were derived from four brains (GFP, DN-AKT), 706 cells, and four control brains (GFP, pcDNA3.0), 540 cells.
For studies of cell identity, histograms of Pax6, Tbr2 and Tbr1 expressing cells were derived from four brains, 690 GFP + cells (Pax6, DN-AKT), four brains, 1099 GFP + cells (Pax6, pcDNA3.0); two brains, 227 GFP + cells (Tbr2, DN-AKT), four brains, 489 GFP + cells (Tbr2, pcDNA3.0); four brains, 652 GFP + cells (Tbr1, DN-AKT), and four brains, 1092 GFP + cells (Tbr1, pcDNA3.0).
Immunohistochemistry and tissue analysis
Brain sections were incubated with blocking solution (5% goat serum and 0.3% Triton X-100 in PBS) for 1 hour at room temperature, and then incubated with primary antibodies diluted in blocking solution for 2 hours at room temperature (for Pax6, Tbr2, Tbr1, mCherry and TOPdGFP) or overnight at 4°C (for Axin2-d2EGFP, phospho-AKT, phospho-GSK3β, phospho-β-catenin, phospho-Vimentin 4A4, and GFP in distribution assay). Primary antibodies used were anti-GFP chicken polyclonal antibody (1:1000, Abcam, 1 Kendall Sq, Cambridge, MA 02139), anti-N-cadherin mouse monoclonal antibody (1:1000, BD Transduction LaboratorieBD Biosciences 2350 Qume Drive San Jose, CA 95131), anti-DsRed (mCherry) rabbit polyclonal antibody (1:1000, Clontech 1290 Terra Bella Ave. Mountain View, CA 94043 USA), anti-Pax6 mouse monoclonal antibody (1:200, Developmental Studies Hybridoma University of Iowa, Department of Biology 028 Biology Building East Iowa City, Iowa 52242–1324), anti-Tbr2 rabbit polyclonal antibody (1:250, Abcam), anti-Tbr1 rabbit polyclonal antibody (1:200, Chemicon EMD Millipore Headquarters 290 Concord Road Billerica, MA 01821), anti-phospho-AKT Ser 473 D9E rabbit monoclonal antibody (1:50, Cell Signaling 3 Trask Lane Danvers, MA 01923), anti-phospho-GSK3β Ser 9 rabbit polycolonal antibody (1:100, Cell Signaling), and anti-phospho-β-catenin Ser 552 rabbit antibody (1:250, kindly provided by Mark Hembree and Linheng Li, Stowers Institute 1000 E 50th St, Kansas City, MO 64110 U). After washing in PBS, the sections were then incubated with secondary antibodies and DAPI diluted in PBS for 1 hour at room temperature. Secondary antibodies used were Alexa488-conjugated goat anti-chicken IgG antibody (1:1000, Molecular Probes Life Technologies 3175 Staley RoadGrand Island, NY 14072 USA), Alexa555-conjugated goat anti-mouse IgG antibody (1:1000; Molecular Probes), Alexa555-conjugated goat anti-rabbit IgG antibody (1:1000; Molecular Probes), Alexa647-conjugated goat anti-mouse IgG antibody (1:1000; Molecular Probes), Alexa647-conjugated goat anti-rabbit IgG antibody (1:1000; Molecular Probes), and Alexa488-conjugated goat anti-mouse IgG antibody (1:1000; Molecular Probes). Finally, immunofluorescent images of the (Ax2GFP; NesCre; NcadFlox/Flox) brains and controls were captured using a Nikon ECLIPSE TE2000-U fluorescent microscope (Nikon Inc. 1300 Walt Whitman Road Melville, NY 11747–3064, U.S.A.). Confocal images were captured using a Zeiss UV LSM510 confocal microscope Carl Zeiss Microscopy, LLC One Zeiss Drive Thornwood, NY 10594 USA). The analysis throughout the paper was carried out in a semi-blinded manner as previously described .
Primary neural stem cell culture, shRNA, and treatment with function-blocking antibodies
For Western blots of N-cadherin, phospho-β-catenin S552, β-catenin, phospho-Akt S473 and Akt, E14.5 mouse primary cortical cultures were generated from E14.5 mouse embryos using minor modifications to the method described in . After dissection, cells were disassociated with 0.25% Trypsin/EDTA (Sigma), washed by DMEM with 10% FBS, and resuspended in clone density media (CDM) containing DMEM (Cellgro Mediatech, Inc. 9345 Discovery Blvd. Manassas, VA 20109), 2 mM L-glutamine (Cellgro), 1 mM N-Acetyl-cysteine, 1 mM sodium pyruvate, and B27 and N2 supplement (final 1×) with 25 ng/mL FGF-2. Five million primary cortical precursors were transfected with 10 µg shRNA constructs against N-cadherin or EGFP control using Nucleofector following the manufacture’s protocol (Lonza Lonza Inc. 90 Boroline Road Allendale, NJ 07401). Transfected cells were plated on Poly-D Lysine treated 6-well tissue culture plates and allowed to recover in the described media (as above) at 37°C for 24 hours before lysing for Western blots. Adherent cortical NSC cultures were generated from E14.5 mouse embryos as described in [30, 31]. After dissection, cells were dissociated with Accutase (Millipore EMD Millipore Headquarters 290 Concord Road Billerica, MA 01821), washed three times with NS-A medium (Stem Cell Technologies #5750 STEMCELL Technologies Inc. 570 West Seventh Avenue, Suite 400, Vancouver, BC, V5Z 1B3, Canada), rinsed once more with NSC medium (NS-A supplemented with FGF-2, EGF, N-2A, B27, L-glutamine, Sodium pyruvate, N-acetyl cysteine, and Penicillin-streptomycin as in ) and plated on laminin-coated (treated with 2 µg/cm2 laminin) dishes. To obtain material for protein analysis, 5 million mouse E13.5 or E14.5 primary cortical precursors was transfected by AMAXA Nucleofection (Amaxa Biosystems, Gaithersburg, MD, USA) following manufacturer protocols; cells were allowed to recover in the described media (as above) at 37°C for 24 hours before lysing for Western blots. Cells were plated at 1.8 × 105/cm2 for 24 hours, then treated with either function-blocking N-cadherin antibody (ACAM, GC-4, Sigma) or control IgG1κ isotype control (BD Pharmingen cat. 554721 BD Biosciences 2350 Qume Drive San Jose, CA 95131) (both at 20 µg/ml, final concentration) for 24 hours.
293 T co-culture and luciferase assays
293 T cells were maintained in cDMEM media containing DMEM (Cellgro), 10% FBS, 2 mM L-glutamine (Cellgro) and Penicillin:streptomycin (Cellgro). For transwell co-culture assays, signaler and reporter cells were transfected separately on day 1 and plated together after 24 hours. On day 1, signaler cells (1 × 106 293 T cells) were transfected with 1.0 µg Wnt or pcDNA3.0 control using Lipofectamine 2000 following the manufacturer’s protocol (Life Technologies Life Technologies 3175 Staley RoadGrand Island, NY 14072 USA) and plated in a 24-well plate. Reporter cells (5 × 105 293 T cells) were transfected with 1.0 µg Super8xTOP FLASH Luciferase and 0.1 µg Super8xFOP Renilla using Lipofectamine 2000, and plated either (1) on the outside of the 6.5 mm transwell membrane inserts with 0.4 µm pore size (Costar Corning Incorporated Life Sciences 836 North Street Building 300 Suite 3401 Tewksbury, MA 01876, USA) inverted in a 6-well plate or (2) on the bottom of a 12-well plate. After 24 hours, the 6.5 mm transwell membrane inserts with reporter cells attached were inverted and placed in a 12-well plate soaked in media. Signaler cells in the 24-well plate were lifted by 0.25% Trypsin/EDTA and one-quarter of the cells were plated on the inside of the transwell membrane inserts (“Membrane-bound” condition). The pore size of these inserts is 0.4 µm, through which cells plated on opposite sides of the insert can establish physical contacts [26, 27]. For the “Separated” condition, the signaler cells were plated inside transwell membrane inserts and placed above the 12-well plate with the reporter cells plated on the bottom of the well . After incubated for 24 hours at 37°C, the reporter cells were lysed for Dual luciferase assay (Dual-Luciferase® Reporter Assay System, Promega Promega Corporation 2800 Woods Hollow Road Madison, WI 53711 USA).
For 293 T co-culture with N-cadherin knockdown, the reporter and signaler cells were generated similarly to that described above (1 × 106 cells were transfected by 1.0 µg Super8xTOP FLASH Luciferase and 0.1 µg Super8xFOP Renilla as reporter cells, and a further 1 × 106 cells were transfected by 1.0 µg Wnt3a or pcDNA3.0 control as signaler cells), with the difference that 0.25 µg N-cadherin/EGFP-shRNA (or NCAD-ΔC/pIRES-GFP control) were transfected into either the reporter or signaler cells; pcDNA3.0 empty vector was used to bring the total DNA mass to 1.6 µg in all the transfections. After 24 hours, the reporter and signaler cells were lifted by 0.25% Trypsin/EDTA, and one-half of the signaler cells and one-sixth of the reporter cells were mixed and plated on a 24-well plate. The cells were lysed after another 48 hours for Dual luciferase assay.
293 T transfection
To collect the protein lysates for N-cadherin knockdown studies, 293 T cells were transiently transfected using Lipofectamine 2000 following the manufacturer’s protocol (Life Technologies). 293 T cells (1 × 106) were plated on a 12-well tissue culture plate and transfected by 1.2 µg Wnt or pcDNA3.0 control together with 2.0 µg N-cadherin or EGFP shRNA construct. Cells were allowed to recover in cDMEM at 37°C for 24 hours before lysing for Western blots. For AKT studies, 1 × 106 293 T cells were plated on a 24-well tissue culture plate and immediately transfected by 1.0 µg HA-AKT-K179M or pcDNA3.0 control.
Western blot analysis
To prepare whole cell lysates, 293 T cells or mouse NSC lines were obtained as described above and collected in RIPA lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS) containing Protease Inhibitor Cocktail Set III (Calbiochem EMD Millipore Headquarters 290 Concord Road Billerica, MA 01821) and Phosphotase Inhibitor Cocktail I (Sigma). Cells were passed through an insulin syringe to shear and centrifuged at 14,000 rpm for 10 minutes at 4°C. Supernatants were collected and combined with 4× SDS sample buffer and boiled for 5 to 10 minutes. The resulting protein samples were applied to 8% or 6% SDS-PAGE gel and transferred to PVDF membranes. The blots were blocked with 5% milk/TBS-T or 5%BSA/TBS-T for 1 hour and probed with the following primary antibodies: rabbit anti-phospho-LRP6 Ser 1490 (1:1000 dilution in 5% BSA; Cell Signaling), rabbit anti-LRP6 (1:1000 dilution in 5% milk; Cell Signaling), mouse anti-N-cadherin (1:1000 dilution in 5% milk; BD Transduction Laboratories), rabbit anti-phospho-β-catenin Ser 552 (1:1000 dilution in 5% BSA; Cell Signaling), rabbit anti-β-catenin (1:1000 dilution in 5% milk; Cell Signaling), rabbit anti-phospho-AKT Ser 473 (1:1000 dilution in 5% BSA; Cell Signaling), rabbit anti-AKT (1:1000 dilution in 5% milk; Cell Signaling), rabbit anti-phospho-GSK3β Ser 9 (1:1000 in 5% BSA; Cell Signaling), rat (rabbit) anti-GSK3β (1:500 dilution in 5% milk; Calbiochem), mouse anti-actin (1:1000; dilution in 5% milk; Chemicon), and rabbit anti-GAPDH (1:1000; dilution in 5% milk; Santa Cruz Biotechnology Santa Cruz Biotechnology, Inc. 10410 Finnell Street Dallas, Texas 75220 U.S.A.). Blots were washed × 3 with TBS-T before incubation with HRP-conjugated goat anti-mouse antibody (1: 3000 dilution in 5% milk, Bio-Rad 2000 Alfred Nobel Drive, Hercules, CA 94547) or HRP-conjugated goat anti-rabbit antibody (1: 5000 dilution in 5% milk, Bio-Rad) for 1 hour at room temperature. The signal was visualized using the ECL Western blotting system (Amersham GE Healthcare Biosciences P.O. Box 643065 Pittsburgh, PA 15264–3065). To ensure equal loading, protein concentrations were determined using the BCA Protein Assay (Pierce Thermo Scientific; 81 Wyman St. Waltham, MA 02454). Densitometry of scanned films was performed with ImageJ.