Figure 2
Confocal images of immunostained sections illustrating developmental changes in cytoarchitecture of organoids. (A–F) 2-week-old organoids were composed of SOX2+/Nestin+ neuronal progenitor cells (NPC) expressing the forebrain marker FOXG1. Formation of first few neuronal rosettes was evident at this stage. (E) (SOX2, Nestin) and (F) (SOX2, FOXG1) show magnified views of the boxed region in (D). Markers of neurons were absent at this stage. (G–L) 4-week-old organoids were in early stages of neurogenesis. The now larger rosettes are composed of a pseudostratified neuroepithelium of SOX2+ NPC surrounding a central lumen. Newborn neurons expressing doublecortin (DCX) and the cortical neuron marker TBR1 surround individual rosettes, often as a thin layer separating individual rosettes. (K) (SOX2, TBR1) and (L) (SOX2, DCX) show magnified views of the boxed region in (J). (M–Q) In 6.5-week-old organoids, the number of TBR1+ neurons is greatly increased and a cortical plate-like zone (CP) has formed in peripheral regions of organoids. At this stage, the majority of TBR1+ neurons co-express the deep layer neuron marker CTIP2. Formation of a distinct subventricular zone (SVZ) separating the ventricular zone (VZ) and the CP is evident. Note the abundant TBR1low newborn neurons located next to SOX2+ progenitors within the SVZ (panel Q shows magnified view of the boxed region in P). Dashed lines in (Q) mark the border between VZ, SVZ and CP. (R–V) Rosettes show hallmarks of a polarized neuroepithelium. The luminal border of the VZ is labelled with apical domain markers (ZO-1, Phalloidin-stained F-actin) throughout organoid differentiation. Images show sections of 2-week- (R,T), 4-week- (S,U) and 7-week-old organoids (V). Apico-basal polarity of the neuroepithelium is also reflected by restriction of cell divisions (arrows in T–V) to the apical border. Scale bars: 200 µm (A–D,G–J,M–P,S,R), 100 µm (E,F,K,L), 50 µm (Q), 20 µm (U–V).
