Figure 1.

Cerebral cortex differentiation and expression analyses. (A): Schematic diagram of the culture procedure for cortical neuron differentiation. (B): Western blotting analysis on day 18. Each WNT inhibitor inhibited Wnt/β-catenin-dependent signaling during the SFEBq differentiation procedure, but C59 also inhibited Wnt/β-catenin independent signaling. (C, D): Time course analyses of the expression of WNT factors show characteristics of a rostral-low and caudal-high gradient. (E): Gene expression analyses of various differentiation markers. The expressions of undifferentiation markers, OCT3/4 and NANOG, were decreased, while the expressions of telencephalon markers, SIX3, EMX1, PAX6, and LHX2, and of the specification marker of corticospinal motor neurons, FEZF2, were increased. (F–H): Time course analyses of the expressions of markers for the telencephalic region. Differences were considered significant by ANOVA and Tukey’s test. ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001; ∗∗∗∗, p < .0001 (n = 4). Abbreviations: bFGF, basic fibroblast growth factor; C59, WNT-C59; DMEM, Dulbecco’s modified Eagle’s medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KSR, Knockout Serum Replacement; OFL, ornithine-fibronectin-laminin; pPKC, phosphorylated PKC; SFEBq, serum-free floating culture of embryoid body-like aggregates with quick reaggregation; XAV, XAV939.