Figure 4.
Dynamic growth cones and somal migration during astrocyte-to-neuron conversion, as observed by two-photon live imaging.
(A) Two-photon time-lapse images illustrating a newly converted neuron (arrowhead) extending a long axon-like neurite with multiple growth cones (white box). Note the retraction and extension of the growth cones (arrowheads) within 24 hours (right insets). The same group of growth cones in the white boxes at 9, 9.5, and 10 dpi is pseudocolored blue, green, and red, respectively, and then overlaid to highlight growth cone dynamics over time. Scale bars: 20 μm, 10 μm (insets). (B) Two-photon time-lapse images showing tangential migration of a lineage-converting cell (arrowhead). The same group of cells infected with the CAG::NeuroD1-P2A-GFP retrovirus at 4, 6, and 8 dpi was pseudocolored blue, red, and green, respectively, and then overlaid to highlight tangential somal translocation over time. A typical non-migrating cell is indicated with an arrow. Scale bar: 50 μm. (C) Two-photon time-lapse images showing vertical migration of a lineage-converting cell after AAV9 GFAP1.6::NeuroD1-P2A-GFP infection. Note the vertical somal translocation (arrowhead) from 11–20 dpi. Original Z-stack images were resliced along the X–Z plane to obtain a lateral view of the mouse cortex (pia/top, ventricle/bottom). Scale bar: 10 μm. (D) Quantification of cell migration velocity after viral infection. Note that the proliferating cells and lineage-traced cells overexpressing NeuroD1 migrated at average velocities of ~8 and ~6 μm/day, respectively. Meanwhile, the proliferating or lineage-traced astrocytes overexpressing GFP alone did not migrate at all. One-way analysis of variance followed by Tukey's post hoc test, mean ± SD, n = 10 animals, ****P < 0.0001, Ftreatment (3, 36) = 72.59. dpi: Day(s) post infection; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; NeuroD1: neuronal differentiation 1.