, 2010), are enriched in postcrossing commissural axons and also increase in a time-dependent manner in vitro. Inhibition of 14-3-3 function switches the response to Shh from repulsion to attraction in vitro and prevents the correct AP turning of postcrossing commissural axons in vivo. Conversely, premature overexpression of 14-3-3 proteins in vitro and in vivo drives the switch in Shh response from attraction to repulsion. 14-3-3 proteins switch the turning response to Shh by reducing PKA activity. Hence, we identify a 14-3-3 protein-dependent

mechanism for a cell-intrinsic time-dependent switch in the polarity of axon selleck inhibitor turning responses. This allows commissural axons, which are first attracted ventrally toward the floorplate by Shh, to switch their response to Shh so that they become repelled by Shh after crossing the floorplate and migrate anteriorly along the longitudinal axis. To evaluate the role of the floorplate and floorplate-derived cues in the migration of postcrossing commissural axons, we analyzed Gli2−/− mouse embryos, which lack a floorplate. In these mutants, commissural axons still project to the midline in response to Netrin-1 in the ventral ventricular zone ( Matise et al., 1999). We used DiI anterograde labeling of commissural axons of E11.5 embryos, shortly after commissural

axons have begun to cross the floorplate, to KU-55933 mouse visualize the trajectory of postcrossing commissural axons. After diffusion of the DiI, the

neural tube was prepared in the open-book format for analysis of the commissural axon trajectories ( Figure 1A). In control Gli2+/− embryos, labeled axons exhibited the stereotypic commissural axon trajectory: most axons migrated ventrally toward the midline, crossed the floorplate, and turned anteriorly ( Figure 1B). In Gli2−/− neural tubes, axons still migrated ventrally to the midline but became severely disorganized at the midline. all Although axons still switched from a DV to an AP axis of migration at the midline, their AP directionality appeared random ( Figure 1B), consistent with previous studies by Matise et al. (1999). Approximately 50% of the total fluorescence of the axons was distributed anteriorly, indicating complete randomization of the AP guidance of axons ( Figure 1C). Thus, whereas the floorplate is not required for axons to switch from a DV to an AP axis of migration, it is required for the axons to correctly turn anteriorly after midline crossing. This suggested that a floorplate-derived cue is important for correct anterior turning of postcrossing commissural axons. One candidate floorplate-derived molecule that could act as a guidance cue along the longitudinal axis is Shh, which attracts precrossing commissural axons ventrally to the floorplate in mammals (Charron et al.