Figure 4. ALI-COs exhibit diverse axon tract morphologies.

a. Staining for all axons (SMI312) and dendrites (MAP2) on an ALI-CO after 36 days at the ALI (85 days total) reveals thick bundles (arrowheads) that can be seen projecting within the organoid and merging to form large tracts (inset below, arrowhead). Representative image of seven ALI-COs stained with similar results. b. Staining for the marker of corpus callosum, NRP1, reveals several internal tracts that are positive and even appear to turn. MAP2 stains dendrites. Age: 49 days at the ALI, 120 days total. Representative image of four ALI-COs stained with similar results. c. Costaining for NRP1 and the callosal guidance factor WNT5A reveals discrete foci (yellow arrows) surrounding an NRP1⁺ internally projecting tract in an ALI-CO at 54 days at the ALI, 117 days total. Image at right is magnification of boxed region. Representative image of two ALI-COs stained. d. Staining for the axon attractant Netrin 1 reveals large areas of positivity with evident tracts (SMI312⁺, arrows) projecting inward and toward the Netrin 1 signal. Age: 32 days at the ALI, 81 days total. Representative image of two ALI-COs stained. e. Axon (SMI312) and dendrite (MAP2) staining of an ALI-CO with tract “escaping” from the main mass (arrows) after 34 days at the ALI (89 days total), in addition to internal projections (arrowheads). Representative image of seven ALI-COs stained with similar results. f. Analysis of axon alignment and coherency by OrientationJ analysis (detailed in methods) of SMI312 staining in a whole ALI-CO at 41 days at the ALI (89 days total). Pixel brightness corresponds to coherency while hue corresponds to energy, where pixels with higher energy report less isotropic and more oriented structures²³. Representative analysis shown out of six ALI-COs analyzed with similar results. Scale bars: 500 μm in a, c, d, e, f, 1 mm in b, 100 μm in c inset, 200 μm in d inset and e inset.