Tissue was rinsed three times in HBSS without Ca2+/Mg2+ (Invitrogen, #14170), incubated for 15 min at 37°C in HBSS without Ca2+/Mg2+ supplemented with 1 mM EDTA, and gently pipetted to obtain a single-cell suspension. Cells (1 × 105) were plated on coverslips coated with ephrin or Eph Fc fusion proteins and routinely cultured in Neurobasal (Invitrogen, 21103) supplemented with 1× N2 (Invitrogen, 17502), 1× B27 without FXR agonist VitA (Invitrogen, #12587), 2 mM L-glutamine (Invitrogen, #25030), and 50 U/ml penicillin/streptomycin

(Invitrogen, #151070). After 1 hr and 24 hr, plates were gently tapped and rinsed with PBS, and cells were fixed in 4% paraformaldehyde for 30 min, washed three times in PBS, and then stained for GFP and Hoechst. At least 500 cells from 5 to 10 random fields per experiment and condition were counted by a blinded person, and the proportion of GFP+ cells among the total number of cells per field was calculated. Brains were in utero electroporated at E13.5 with the indicated plasmids. At E14.5, electroporated cortices (n = at least three per condition) were dissected in cold

HBSS and lysed in NP40 buffer (containing 20 mM Tris-HCl at pH 8, 137 mM NaCl, 10% glycerol, 1% Igepal [NP-40], 2 mM EDTA). Extracts were cleared by centrifugation for 15 min at 4°C and precleared for 1 hr with protein A/G beads. Immunoprecipitation was performed with check details 2 μg of anti-ephrin-B1 (R&D Systems), anti-NeuroD1 (control antibody, Santa Cruz Biotechnology),

or anti-MYC (Roche) overnight at 4°C, followed by incubation for 1 hr with protein A/G beads; washing and elution were performed according to standard protocols with NP40 buffer. Western blotting was performed according to standard protocols with antibodies recognizing ephrin-B1, GFP, Myc, and Actin. We thank Gilbert Vassart for continuous support and interest; members of the lab and the Institut de Recherche en Biologie Humaine et Moléculaire for helpful discussions and advice; Dr. Bollet-Quivogne (Fonds de la Recherche Scientifique [FNRS] Logistic Scientist) of the Light Microscopy Facility for his support with imaging; Giuseppe Saldi for computation of the time-lapse analysis before data set; and Viviane De Maertelaer and Jerome Bonnefont for advice on statistical analyses. We thank Dr. Hoshino and Dr. Collard for reagents to study P-Rex1 and Rac3. This work was funded by grants from the Belgian FNRS, Fonds pour la Recherche de l’Industrie et l’Agriculture, and Fonds pour la Recherche Scientifique Médicale; the Belgian Queen Elizabeth Medical Foundation; the Action de Recherches Concertées Programs; the Interuniversity Attraction Poles Program; the Belgian State; the Federal Office for Scientific, Technical and Cultural Affairs; the Welbio and Programme d’Excellence CIBLES of the Walloon Region; the Fondations Université Libre de Bruxelles; and Pierre Clerdent and Roger de Spoelberch (to P.V.). P.V. is research director, L.T.