Fig. 6: Characterization of the neural plate and formation of the neural tube.

a–c Folding of the neural plate (fnp), nf: neural fold. AP and DV axes are indicated. b, c Expression of Sox2 seen by b immunostaining (number embryoid N = 4/4), c in situ hybridization (N = 2/2) on transverse section. d Front view of an embryoid showing the folded neural plate closing into a neural tube, dorso-lateral hinge points (dlhp). e–h Single or double colour in situ hybridization for e Olig2 revealing motor neuron progenitor (mnp). f Shh (red) in the notochord and floor plate (n/fp) in a territory immediately ventral to Olig2 (blue) expression domain. g, h Expression of Sox10 revealing the neural crest cells (nc) in dorsal view (N = 2). h Transverse section of an embryoid after double colour in situ hybridization for Sox2 (red—neural plate) and Sox10 (blue—neural crest cells). i–m Transverse sections of a folding neural plate (N = 9/9). j The organization of a pseudostratified columnar epithelium. Green: GFP cells extending from the apical to the basal side of the epithelium (N = 3/3) k apical (ap)–basal (ba) polarity of the epithelium with apical accumulation of actin labelled with Phalloidin and basal accumulation of laminin (N = 3/3). l Immunolabelling for Phospho-Histone H3 (PHH3) revealing that cell divisions occur at the apical side of the epithelium (number of embryoids N = 3/3, number of mitotic cells at the apical side N = 7/8). m Immunolabelling revealing the presence of neuronal cell bodies (n) expressing TUBB3 at the basal side of the neural plate (N = 3/3). n, o Expression of TUBB3 (red) showing neurons extending their axons in the neural plate and toward the non-neural part of the embryoids (n, lateral view; o, dorsal view of the same embryoid). p TUBB3 expressing neurons (red) in multiple layers of the anterior neural plate in a parasagittal section at D9 (Sox1-GFP, green) (N = 2/2). q, r Expression of DCX (red) confirming the presence of neurons and axons extending both within the neural plate and toward the non-neural part of the embryoids (N = 4/4). s Neural plate closed into a neural tube (N = 2/8). g–s Hoechst is labelling the nuclei. Developmental stages of the embryoids and drawing of embryos bottom right of each panel. Scale bars: 100 µm. Numbers of performed experiments of biological samples analysed and variability in genes expression are provided in Supplementary Fig. 14.