All animal protocols were approved by the UCSF IACUC. P5 CD1s (Charles River) were used for cultures. BAT-gal mice (Maretto et al., 2003
) were used for β-galactosidase (β-gal) expression analysis and for DNLef1 electroporations. C57Bl6 mice (Jackson) were used for Dkk1 electroporations. Males and females were used for all experiments.
pLNCX2 (Clontech) was modified to include a new multiple cloning site and histone H2B fused to monomeric red fluorescent protein (pLH2BmRFP). A dominant-negative Lef1 (DNLef1) construct (Lef1 (Clontech) with a truncated N-terminus) was inserted. Retrovirus was produced in GP2–293 cells (Clontech) and concentrated using standard methods.
In utero electroporations (EP)
See (Li et al., 2008
) for method. EPs were targeted to the lateral cortex at E13.5 or E15.5 and collected 4 or 6 days later. The DNLef1 construct was inserted into pCAGGS (chicken β-actin cytomegalovirus promoter-driven expression). pCIG (GFP) (from A. McMahon (Megason and McMahon, 2002
)) was used as a control. pCIG-Dickkopf-1 was provided by L.W. Burrus.
Immunohistochemistry and X-Gal staining
Brains were processed using standard methods. Cells and tissue sections were stained using standard protocols with: rat anti-PDGFRα (BD Biosciences Pharmingen,1:500 for tissue and 1:800 for cells), rabbit anti-Olig2 (Millipore, 1:1000 for tissue), mouse anti-β-gal (Promega, 1:2000 for tissue and 1:6000 for cells), and mouse anti-O4 (Sigma, 1:30 for cells). Fluorescent secondary antibodies (1:1000, Invitrogen) and DAPI (1:3000, Sigma) were used. For X-gal staining, floating sections were stained with X-gal substrate (Invitrogen).
Neural progenitor cells (NPCs) were from lateral ventricle SVZ tissue of P5 CD1s. Tissue was dissociated with 0.1% Trypsin (Worthington) and 0.1% DNAse1 (Roche). DMEM/F12 (50:50, Gibco) was supplemented with 10X hormone mix (40 mg transferrin, 10 mg insulin, 3.86 mg putrescine, 4.0 ml 3mM selenium, 4.0 ml 2mM progesterone, 10 ml 2 mg/ml Heparin [all from Sigma]), 0.8 ml 30% glucose, 0.6 ml 7.5% NaHCO3, 10 ml 30% glucose, 7.5 ml 7.5% NaHCO3, 2.5 ml 1 M HEPES, 5 ml 200 mM glutamine, 5 ml Pencillin-Streptomycin, 2 ml Fungizone). Media containing EGF (Sigma, 10 ng/ml), FGF (Sigma, 20 ng/ml) and B27 (Invitrogen) was added. Cells were plated at 80,000 cells per 25 cm2 flask (Corning), grown in 5 ml of complete media plus growth factors and B27. Passage 1 or 2 NPCs were used.
Cell culture – Wnt treatments
NPCs were dissociated with 0.05% trypsin-EDTA, washed with PBS, pelleted and resuspended in 500µl of complete media with 2% FBS. The cells were plated on laminin-coated (1 mg/ml, Invitrogen) chamber slides (Nunc) at 37,500 cells/well. Mouse Wnt3a (0.15ng/µL), Dickkopf1 (Dkk1, 0.03ng/µL), or an equivalent volume of PBS were added to each well. One half of the media was replaced each day. The cultures were analyzed after 4 days in vitro (DIV).
Cell culture – Retroviral infection
NPCs were dissociated, washed with PBS, pelleted and resuspended in 500µl complete media containing EGF, FGF, B27 and 5 µg/ml polybrene (Sigma). Concentrated virus (50–100 µl; approx 1×105 transducing units) was added to approximately 300,000 cells, which were spin infected for 90–120 min at 170 × g. The cells were plated in complete media containing growth factors in a 25cm2 flask for 24 h to allow integration and expression. Cells were resuspended in complete media with 2%FBS and plated on chamber slides at 37,500 cells/well and analyzed after 4 DIV.
Image analysis and quantification
Three to four biological replicates of each experiment were performed. Ten 20X magnification images were quantified for each replicate of each cell culture condition. Cell counts from 3–4 coronal sections of each EP brain were averaged.
Results are expressed as mean ± SEM. Data were analyzed using two-tailed Student’s t test with unequal variance. Multiple comparisons were made using ANOVA with a post-hoc Holm-Sidac test. Any value of p ≤ 0.05 was considered significant.