Modeling Human Interneuron Migration using hfMCOs(A) Scheme illustrating the strategy of organoids fusion for modeling human interneuron migration.(B) Images showing hsyn-RFP labeled MGE progenitors (arrows) migrated towards hCO sideduring culture. dpf: days post fusion. Scale bar, 10 μm.(C) 3-D reconstruction of hfMCOs revealed the migration of NKX2-1-GFP+ progenitors in hCO side. Z-stack con-focal imaging was performed near the fusion border. Note that cells have already started to migrate out 3 dpf (arrows). Scale bar, 10 μm.(D and E) Immunostaining for GFP in hfMCOs section 21 dpf confirmed the migration of MGE progenitors. Arrows show migration directions. Scale bar, 40 μm.(F and G) Representative images showing typical forward movement of growth cone (F, arrows) and soma translocation observed for migrating NKX2-1-GFP+ progenitors (G, arrows) at 14 dpf. Yellow box: cytoplasm elongation proceeding nucleokinesis. White box: neurons that migrated out of the focal plane. Scale bar, 10 μm.(H) Representative images showing migration directions of NKX2-1-GFP+ progenitors in hfMCOs without (left) or with 50 μM blebbistatin treatment (right). Yellow arrows: migration directions. Yellow stars: neurons without migration detected. Scale bar, 10 μm.(I-K) Quantification of ratio of migrating neurons (I), migrating speed (J), and ratio of active growth cones (K). Mean ± SD are plotted. **, P<0.01; ***, P<0.001.(L) Representative images of random movement of growth cone in the presence of 50 μM blebbistatin. Yellow arrows: movement directions. Scale bar, 5 μm.(M) Representative image showing hsyn-GCaMP6s-expressing interneuron migrated into RFP-labeled hCO in an intact hfMCO at 12 dpf (left), the spontaneous calcium surges (middle), and the quantification (right). Mean ± SD are plotted (n=6, totally 85 cells). Scale bar, 20 μm.(N) Immunostaining for vGAT and GFP in section of hfMCO. Arrow indicates the migration direction. Scale bar, 20 μm.(O) Immunostaining for PSD95 and GFP in section of hfMCO. Arrow indicates the migration direction. Scale bar, 5 μm.See also Figure S6-S7; Movie S4-S6.
