R18 increased the percentage of cohorts with randomization of AP guidance along the longitudinal axis (p = 0.0041), but there was no significant effect on the amount of stalling or recrossing axons (Figure 6B). When we quantified the degree of AP randomization by measuring the fraction of

anterior fluorescence, there was a significant decrease in the amount of anterior axons in the presence of R18 (Figure 6C). The decrease was in between that observed for the Wnt1-Cre;Smon/c and Math1-Cre;Smon/c mice. To inhibit 14-3-3 activity specifically in neurons, we turned to the developing chick embryo. Plasmids encoding R18 or a control WLRL peptide fused to EGFP MS-275 research buy (Kent et al., 2010) were injected into chick neural tubes and electroporated unilaterally. Two days later, the embryos were dissected and the commissural axon trajectories analyzed in the open-book format. Electroporated neurons were

identified by EGFP expression. In neurons expressing control WLRL-EGFP, the vast majority (92%) of axons correctly made an anterior turn after crossing the floorplate, with very few axons making a posterior turn. In contrast, neurons expressing R18-EGFP had a different distribution of phenotypes, with significantly fewer axons (58%) making the correct anterior turn (p < 0.001) and significantly more axons (35%) turning posteriorly (p < 0.001) (Figure 6D). Notably, as with the Tat-R18-YFP-treated rat open-book cultures (Figures

6A and 6B), there was no significant difference in the percentage of CHIR-99021 molecular weight axons stalled in the floorplate or after floorplate exit, indicating that 14-3-3 activity is required Dichloromethane dehalogenase for AP guidance, but not floorplate crossing and exit. This is different than what we observed in the Wnt1-Cre;Smon/c and Math1-Cre;Smon/c mice, which have defects in both AP guidance and floorplate crossing and exit. Together with our in vitro data demonstrating that 14-3-3 activity is required for the switch in Shh response from attraction to repulsion, this implicates 14-3-3 proteins in mediating the repulsive response to Shh in postcrossing commissural axons. If 14-3-3 proteins are key mediators of the switch in the polarity of the turning response, overexpression of 14-3-3 proteins should be sufficient to prematurely activate the switching program in young neurons. We tested this in vitro by transducing dissociated commissural neurons with HSV (herpes simplex virus) expressing either 14-3-3β or 14-3-3γ, the two 14-3-3 isoforms that are enriched in postcrossing commissural axons. Control neurons transduced with GFP alone were attracted up a Shh gradient at 2 DIV (mean angle turned of 18.0° ± 5.1°) (Figures 7A and 7B). Expression of either 14-3-3β or 14-3-γ significantly changed the turning response of the neurons to a Shh gradient. Overexpression of 14-3-3β silenced the attractive response to Shh (mean angle turned of −2.4° ± 4.2°).