In vertebrate embryos, neural crest cells migrate only through the anterior half of each somite while avoiding the posterior half. We demonstrate that neural crest cells express the receptor neuropilin 2 (Npn2), while its repulsive ligand semaphorin 3F (Sema3f) is restricted to the posterior-half somite. In Npn2 and Sema3f mutant mice, neural crest cells lose their segmental migration pattern and instead migrate as a uniform sheet, although somite polarity itself remains unchanged. Furthermore, Npn2 is cell autonomously required for neural crest cells to avoid Sema3f in vitro. These data show that Npn2/Sema3f signaling guides neural crest migration through the somite. Interestingly, neural crest cells still condense into segmentally arranged dorsal root ganglia in Npn2 nulls, suggesting that segmental neural crest migration and segmentation of the peripheral nervous system are separable processes.KEY WORDS: Trunk neural crest migration, Sclerotome, Neuropilin 2, Semaphorin 3F, Mouse, Chick Development 133, 99-106 doi:10.1242 DEVELOPMENT 100 performed in 50% formamide, 1.3ϫ SSC (pH 5), 5 mM EDTA, 50 g/ml yeast RNA, 0.2% Tween 20, 0.5% CHAPS and 50 g/ml heparin at 70°C. Embryos were washed twice in hybridization mix at 70°C, three times in wash solution I at 65°C, and antibody pre-treatment was performed in 100 mM maleic acid, 150 mM NaCl, 0.1% Tween (pH 7.5) with 2% Blocking Reagent (Boehringer Mannheim). Templates for digoxigenin-labeled antisense riboprobes were as follows: chick Npn2 (Gammill and BronnerFraser, 2002), mouse Npn2 (Giger et al., 2000), Sox10 (Kuhlbrodt et al., 1998), Sema3f (Giger et al., 2000), ephrinB2 (Wang and Anderson, 1997), Tbx18 (Kraus et al., 2001) and Uncx4.1 (Mansouri et al., 1997). Stained embryos were infiltrated with 5% sucrose, 15% sucrose and 7.5% gelatin in 15% sucrose, frozen in liquid nitrogen, sectioned at 20 M by cryostat (Microm) and mounted in permafluor (Thermo Electron Corporation).
ImmunohistochemistryNeural crest cells with were stained with 1:50 anti-HNK-1 (American Type Culture; Tucker et al., 1984) followed by 1:400 anti-mouse-IgM-Rhodamine Red X (Jackson Immuno Research) or 1:2000 anti-p75 (Weskamp and Reichardt, 1991) followed by 1:400 anti-rabbit-Rhodamine Red X (Jackson Immuno Research). Sema3f spots were visualized using an anti-mouse IgG Alexa 488 secondary at 1:1000 (Molecular Probes). Unstained embryos were infiltrated with 5% sucrose, 15% sucrose and 7.5% gelatin in 15% sucrose, frozen in liquid nitrogen, sectioned at 15 M by cryostat (Microm) and degelatinized for 20 minutes at 42°C in PBS. Dorsal root ganglia were stained with 1:500 anti-TUJ1 (neuron specific class III -tubulin; Babco) followed by 1:500 anti-mouse-Biotin (Jackson Immuno Research), and developed using the ABC-horseradish peroxidase kit (Vector Laboratories) and 0.1 mg/ml 3,3Ј-diaminobenzidine tetrahydrochloride (DAB; Sigma) with 0.009% hydrogen peroxide according to the manufacturer's instructions.
Conditioned medium293T cells were transfected with 24 g of AP-Sema3f (Giger et al., 2000) o...