Supplementary Figure 4: ALI–COs exhibit both intracortical and escaping tract morphologies with specific patterns of guidance cues.
From: Cerebral organoids at the air–liquid interface generate diverse nerve tracts with functional output
a. Costaining of NRP1 and SMI312 reveals a specific subset of tracts that are positive for NRP1 suggesting callosal identity. Age: 55 days at the ALI, 147 days total. Representative image of four ALI-COs stained with similar results. b. Costaining for NRP1 and RYK reveals a subset of axons with a putative callosal identity expressing the WNT receptor RYK. Age: 19 days at the ALI, 84 days total. c. Staining of labeled tracts (fGFP+) for EphrinB1 demonstrates the presence of internally projecting axons that are also positive for this receptor (arrow). Age: 19 days at the ALI, 84 days total. b-c are representative images of three independent experiments. d. Axon (SMI312) and dendrite (MAP2) staining of an independent ALI-CO (34 days at the ALI, 89 days total) also exhibits the presence of tracts projecting away (magnified inset) from the main mass. Representative image of seven ALI-COs stained with similar results. e. Retrograde labeling with DiI crystals (black arrow, site of injection) of an escaping tract reveals the morphology of the tract (white arrow) and the location of projecting cells (magnified inset, arrowheads) within the ALI-CO (57 days at the ALI, 149 days total). DiI labeling was successful in one ALI-CO shown here. Scale bars, 200 μm (a, d inset), 100 μm (b, a inset, c inset, e inset), 50 μm (b inset) and 500 μm (c, d).
