Figure 4. enCORs display radial units and radial neuronal migration.

a. Nuclear staining by DAPI in a 60-day H9 enCOR reveals radially organized units (dashed lines) while sparse staining for radial glial fibers with the marker of mitotic radial glia phosphorylated vimentin reveals fibers that extend the width of the tissue (arrowheads) reminiscent of a radial scaffold. b. Nestin staining for radial glia in day-70 H1 enCORs reveals long basal processes (arrowheads) that terminate with end feet at the surface of the organoid (asterisks) outside the CP and MZ. c. Electroporation of the VZ of a 64-day H9 enCOR with a GFP construct and vibratome sectioning the next day reveals individual RG basal processes (arrows) that extend to the outer surface (asterisks). d. Frames from live imaging of an outer/basal RG (arrow marks the cell body) in a H1 enCOR electroporated with GFP on day 63, followed by vibratome sectioning four days later and live imaging 24-hours later (Supplementary Video 1). Note the long basal process (arrowheads) and endfoot (asterisk) as well as a division event including mitotic somal translocation beginning at 21:40. The newly generated daughter cell (blue arrow) then extends a process apically (blue arrowheads). Time stamp is hours:minutes. e. Frames from live imaging (Supplementary Video 5) of a membrane targeted farnesyl-GFP labeled neuron (arrowhead) showing radial migration into the CP. Time stamp is hours:minutes. f. Live imaging of migration of several neurons (arrowheads) in the same sample as d., displaying typical radial migration including transient stalling with multipolar morphology (for example blue arrowhead 11:40 to 23:00). Stills taken from Supplementary Video 1. Colors of the arrowheads match traces shown in g. and h. Time stamp is hours:minutes. g. Traces of distance traveled of 22 individual neurons from Supplementary Videos 1, 2, and 4. Colors of the traces match those shown in h. Note the characteristic transient stalling in several of the traces (red, blue, purple) with shorter duration stalling in the green and brown traces, while the light blue trace is already stalled at the start of the movie. h. Movement traces of individual neurons quantified in g. and shown in f. showing the movement into the intermediate zone with stalling and subsequent movement into the CP. i. Individual neurons labeled by electroporation of an H9 enCOR at day 64 with an integrating farnesylated GFP construct to allow for long-term labeling and analysis after 36 days. Note the primary dendrite extending from the cell body (arrows) toward the outer surface (dashed lines), as well as many parallel fibers (arrowheads) in the outer MZ. Scale bars: 100 μm in a., b., c., h., 50 μm in d., f., 20 μm in e., i.