Fig. 3

Confocal imaging of cryo-sectioned day 17 hCSs reveals incomplete acquisition of neural rosette 3D morphology. a–d Representative confocal images of sectioned (20 µm) day 17 hCSs; a ZO-1 staining of apical membrane is surrounded by apico-basal polarised Nestin filaments; b Pax6-positive cells self-organise around N-cad positive foci; c Immature DCX-positive neuronal processes are found outside of neural rosette structures; and d Map2-positive neuronal processes are also found to surround Pax6-positive NPCs organised into rosettes. e Confocal imaging of 60 μm sectioned day 17 hCSs express signs of cortical rosette formation. Newly formed PKCλ-positive membranes are seen at the edge of the tissue as indicated by yellow arrows. f Within thicker sections (60 μm), internalised tubular structures positive for N-cad are observed, suggesting that the inner lumen of neural rosettes may form into tube-like structures. g Phalloidin 488-positive actin filaments (RDYPROBE) show a concentrated organisation around ventricle-like structures. Magenta arrows in e–g images highlight the ovoid formation of cortical rosettes with a pinched end, whereas blue arrows highlight formation of a tubular-like structure. Scale bars = 100 µm (a–d); 50 μm (e–g)