2002; Rakic 2003) and an important guide for migrating cortical neurons (Rakic 1972; ORourke et al. out or blocked with antibodies. Antibodies blocking NgR or Lingo-1 showed the same motility-enhancing effect supporting a direct role of surface Nogo-A on migration. Bromodeoxyuridine (BrdU) labeling of embryonic day (E)15.5 embryos demonstrated that Nogo-A influences the radial migration of neuronal precursors. At E17.5, Rabbit Polyclonal to RNF149 the normal transient accumulation of radially migrating precursors within the subventricular zone was not detectable in the Nogo-A KO mouse cortex. At E19, migration to the upper cortical layers was disturbed. These findings suggest that Nogo-A and its receptor complex play a role in the interplay of adhesive and repulsive cell interactions in radial migration during cortical Vanoxerine 2HCl (GBR-12909) development. 0.05. Results Nogo-A Is Expressed in Radial Glial Cells, Migrating Postmitotic as well as Postmigratory Neurons of the Embryonic Mouse Cortex The Nogo-A expression pattern was assessed during forebrain development in mouse embryos. Nogo-A+ cells were detected in all cortical layers at E15.5 and E17.5 (Fig. 1and Supplementary Fig. S3). In contrast, TROY was only present in nestin+ cells and not in TubIII+ immature neurons. When live cultures were stained with the anti-Nogo-A antibody, we found that Nogo-A was distributed in a punctate manner on the surface of the cells, similar to previous observations on dorsal root ganglion neurons (Dodd et al 2005) and oligodendrocytes (Oertle et al. 2003) (Fig. 2= 70) compared with that of WT cells (64.45 m 3.21/7 h, = 81; Fig. 3= 74) compared with control antibodyCtreated WT cultures (64.66 m 4.83/7 h, = 104; Fig. 3= 81) and Nogo-ACdeficient cells (0.654 m 0.036/min, = 70; Fig. 3= 74) than that of cells with control antibody treatment Vanoxerine 2HCl (GBR-12909) (0.594 m 0.033/min, = 104). In addition, we found that Nogo-ACdeficient cells paused less frequently (0.914 pauses 0.128/7 h, = 70) than WT cells (1.346 pauses 0.161/7 h, = 81; Fig. 3= 104; anti-Nogo-A: 0.892 pauses 0.106/7 h, = 74; Fig. 3= 71; Nogo-A KO: = 72); ** 0.01 and * 0.05, unpaired Students = 55; anti-Lingo-1: = 57; anti-NgR: = 60; and control IgG: = 154); ** 0.01 and * 0.05, analysis of variance, Tukeys test. Scale Vanoxerine 2HCl (GBR-12909) bar = 100 m. At the time of the video analysis, that is, 12 h after plating of the neurospheres on PDLCcoated dishes, the large majority of the migrating cells were nestin+. We then analyzed the differentiation of WT and Nogo-A KO cells after 7 days in differentiation medium by immunocytochemical stainings for neurons, astrocytes, and oligodendrocytes. The same proportions of all 3 CNS cell types were found in both WT and Nogo-A KO cultures: The cultures generated approximately 65% astrocytes (GFAP+), 20% neurons (TubIII+), and 15% oligodendrocytes (APC+; Supplementary Fig. S4). The same proportions of astrocytes, neurons, and oligodendrocytes were also found in cultures of WT cells grown in the presence of the anti-Nogo-A antibody 11C7 or of a control antibody. Since no significant difference in fate specification was found when Nogo-A was knocked out or neutralized, we assume that the effects of Nogo-A on cell locomotion are not due to a change in the proportion of differentiated cells with different mobility during the period of live imaging. As described above, NgR and Lingo-1, 2 Nogo receptor components known to be involved in Nogo-mediated growth cone collapse and nerve fiber growth arrest (GrandPre et al. 2000; Oertle et al. 2003; Mi et al. 2004), were present in the nestin+ neurosphereCderived cells (Fig. 2 0.001 and ** 0.01, unpaired Students and and 0.05, unpaired Students = 0.13; Fig. 5 em J /em ). Discussion Myelin-derived Nogo-A is one of the major inhibitory molecules for axon outgrowth in the adult CNS. While it has been intensively studied in this context, its function in neurons, where it is prominently expressed during development, still remains unclear. The present results suggest that Nogo-A plays a Vanoxerine 2HCl (GBR-12909) role for the radial migration of cortical precursor cells: In vitro, surface Nogo-A negatively modulated the locomotion of precursor cells via the Nogo receptor constituents NgR and Lingo-1, and in vivo, the radial migration of neuronal precursors in the E15C19 forebrain was disturbed in Nogo-A KO mice. During cortical development, Nogo-A is present in and on the surface of migrating and postmigratory neurons and in and on radial glial cells (Mingorance-Le Meur et al. 2007), a major source of neurons and glia Vanoxerine 2HCl (GBR-12909) (Gotz et al. 2002; Noctor et al. 2002; Rakic 2003) and an important guide for migrating cortical neurons (Rakic 1972; ORourke.