Axonogenesis and synaptogenesis in hESC-derived retinal tissue. (a, a′) Immunostaining of hESC-derived retinal tissue with antibodies to NF200 (mature axonal marker) and human Synaptophysin (Syn). DAPI-stained nucleus. Scale bar: 20 μm. (b, c, c′) NF200 [+] axons outgrowing from the basal side, traversing the retinal tissue, and running along the apical side of hESC-derived retinal section (purple dots). Scale bar: 50 μm. (d) Magnified area shown in b (*) and shows the presence of Synaptophysin [+] cells in the basal layer. (e, f, f′) Confocal images demonstrating the presence of many Synaptophysin [+] synaptic boutons in the apical side. Double asterisk in e marks a group of Synaptophysin [+] cells depicted in f, magnified image. Scale bar: 20 μm. Arrows point to multiple Synaptophysin [+] boutons. (f) A confocal (z-stack) image of two cells in e. Scale bar: 20 μm. (f′) Confocal (z-stack) image of the area marked with white arrows in f. The stack image was virtually resectioned at the x and y planes to demonstrate the presence of individual synaptic boutons.