An index of 0 was attributed to the cultures showing no preferential growth. We prepared 20 μm cryosections covering the entire spinal cord from embryos fixed in 4% paraformaldehyde, embedded in 7.5% gelatin, 15% sucrose in PBS, and incubated over night at 4°C with the following antibodies: Plexin-A1 (1/100, AbCAM), Neurofilament 160 kDa (1/100, RMO Zymed), Ngn1 (1/100, Santa-Cruz), Robo3 (1/100, R&D), DCC (1/100, BD), GFRα1 (1/100, R&D), PSA-NCAM (1/100, DSHB), and secondary antibodies Alexa 594, Alexa 488 (1/500, Invitrogen), and Fluoroprobe 546 (1/100) with bisbenzimide (1/2,000, Promega). For lacZ

staining, spinal cord open books were Androgen Receptor Antagonist prepared, fixed in 4% PFA, and incubated with 5 mM Ferro/Ferri cyanide, 2 mM MgCl2, and

1 mg/ml X-Gal in PBS at 37°C and the reaction was stopped in PBS. Chromogenic immunostaining and in situ hybridization was performed as described in Moret et al. (2007). Nuclei were stained with bisbenzimide (Promega) and actin with TRITC-phalloidin in neuronal cultures. Spinal cords were dissected from E12.5 and E13.5 embryos of the gdnf NrCAM, NrCAM/gdnf, NCAM, and RET-wnt-flox Ibrutinib mouse lines and prepared in an “open book” conformation and fixed in 4% PFA overnight. Small crystals of DiI (Invitrogen) were inserted in the dorsal part of one hemicord. Axon trajectories were observed using fluorescence microscopy after 48 hr. Isolated dorsal spinal cord fresh tissue was incubated for 1 hr at 37°C with control, FPcm, or gdnf and treated according to manufacturer’s instructions (Calbiochem). Calpain activity was measured by fluorogenic activity (Victor 3 multilabel counter, Perkin Elmer). For t-BOC assays, dorsal spinal over cord tissues from E12.5 embryos were dissociated, and cells were plated into polylysin- and laminin-coated glass coverslips in Neurobasal medium (GIBCO) supplemented with B27 (GIBCO), glutamine (GIBCO), and Netrin-1 (R&D) medium.

After 2 days in culture, neurons were incubated with control, FPcm, or gdnf for 1 hr at 37°C. Neurons were then treated with t-BOC (10 μM; Invitrogen) for 30 min at 37°C; staining was observed immediately over 20 min maximum. For the analysis, images from all conditions were collected with the same settings. Using ImageJ, a constant threshold was applied to all images to collect the high t-BOC cell population over the whole population, which was quantified in phase contrast. To measure Plexin-A1 levels, we treated neuronal cultures with control, FPcm, or gdnf and processed for immunohistochemistry with anti-Plexin-A1 antibody and phalloidin. Images of individual neurons were taken and the Plexin-A1 fluorescence was quantified using ImageJ software. To measure Plexin-A1 levels in vivo, transverse sections were performed and processed for immunohistochemistry with anti-Plexin-A1 and anti-Nf160kD antibodies. The fluorescence was quantified using ImageJ software into the FP and the two adjacent PC domains and normalized to the selected area.