Live images were captured in hfMCOs on the hCO side 2 weeks after fusion using the Leica TCS SP5 confocal microscope with 63× objective. The microscope was equipped with a controlled cell culture chamber, which was set up to keep 37°C temp erature and 5% CO2 in the environment during live imaging. A x, y, z, t scanning mode was used to obtain z-stack images for 10-12 hours with 10 min time interval. 4-D reconstruction of the images was then performed using Leica LAS-X software. For myosin II inhibition assay, hfMCOs that showed efficient migration of NKX2-1-GFP+ cells on hCO side were treated with 50 μM blebbistatin, equilibrated for 30 min in 37° spinning culture incubator with 5% CO2, and then live imaging was performed as described above. Quantification of neuron migration was performed using Leica LAS-X software. Only focus planes clearly capturing somas were used, and migrating speed was calculated as follow: speed=distance of soma translocation (mm)/[(F2-F1)×10 (min) [, in which F2 and F1 represented the frame number after and before soma translocation happened. % of migrating neurons was
