Figure 3. Neurons of ALI-COs exhibit dynamic axon guidance behaviors.

a. Schematic of electroporation and preparation of ALI-COs for live imaging. Inset image shows an organoid after plasmid injection along with a blue dye (FastGreen) to visualize the injected ventricle (arrow). b. Temporal projection image pseudocolored by time (Supplementary Video 2) of an electroporated ALI-CO 5 days after placement at the ALI (69 days total age) showing dynamic axon outgrowth with growth cone retraction (blue arrow to red arrow in the higher magnification inset). Representative image shown out of 5 similarly staged and live imaged ALI-COs. c. Temporal projection image (Supplementary Video 3) after 9 days at the ALI (73 days total age) showing more directed axon outgrowth with progressive extension (blue arrow to red arrow in higher magnification insets). Representative image shown out of 3 similarly staged and live imaged ALI-COs. d. Tracing of individual growth cones over time from ALI-COs at the ALI for 2-5 days (early) and 14-24 days (late) reveals disparate behaviors and velocities (see Supplementary Videos 1, 4). Growth cones in later, established tracts exhibit a higher velocity (bold blue and red lines are average linear regression for each set of data), while earlier growth cones exhibit dynamic retractions (visible in a highlighted trace in purple compared with later highlighted in green). An example image of each is shown above with superimposed traces. Tracing was done on 9 growth cones (early) and 12 growth cones (late) from four organoids generated from two independent batches. e. Axon tracts after 18 days ALI culture (88 days total) demonstrating numerous dense bundles (arrows) with nonrandom projection pattern. Note the same overall pattern but with reinforced bundles compared with Supplementary Fig. 3d, which was taken 4 days earlier. Shown is a representative ALI-CO out of 7 similarly staged and imaged ALI-COs. f. Directional image analysis for the orientation (hue) and coherency (brightness) of aligned axon tracts (original image Supplementary Fig. 3e). Representative image shown out of 5 such directional analyses performed with similar results. g. Still image (left) and kymograph (right) of an extending tract (boxed region) (Supplementary Video 8). Note the higher velocity (shallow slope, dotted line) of incoming growth cones while the leading edge of the tract as a whole progresses more slowly (steep slope, dashed line). Representative results out of three experiments performed. Scale bars: 1 mm in a, 100 μm in b, c, d, e, g; 500 μm in f