Figure 4.

Rho–Rho-kinase signaling in the cortical neuron is required for axon formation at the opposite side from the Nestin-positive cell-contacting neurite. A, Schematic of the screening assay for inhibitors. Nestin-positive cells (red) and cortical neurons (green) were cocultured for 4 h, and the cells were subsequently incubated with the indicated inhibitors for 44 h. B, D, F, Quantitative analysis of the direction of axon formation under various experimental conditions. **p < 0.01, Student's t test. n.s., Not significant. Error bars represent the SEM. n ≥ 40 Nestin-positive cell-interacting neurons. C, Strategy to perturb the function of Rho in cortical neurons. Dissociated cortical neurons (green) were transfected with the indicated plasmids using in vitro electroporation and cocultured with purified radial glial cells (red) for 48 h. TF, transfect. E, Strategy to perturb the function of Rho-kinase in cortical neurons. Cortical neurons (green) were microdissected from E15.5 brains electroporated at E13.5 with the indicated plasmids and subsequently cocultured with purified radial glial cells for 48 h.