Figure 2.

Notch inhibition of PAX6⁺ progenitors generates TBR1⁺, early-born cortical neurons

(A) Under basal conditions, FACS-isolated progenitors generated PAX6-mCherry⁺/SOX2⁺ rosettes, which persisted for >7 days and showed few MAP2⁺ neurons.

(B) DAPT treatment downregulated mCherry, rapidly generated neurons, and depleted SOX2⁺ progenitors.

(C and D) Quantification for MAP2⁺ (C) neurons or SOX2⁺ (D) progenitors ± DAPT (n = 3 independent experiments).

(E) DAPT had no effect on NOTCH1 but decreased downstream HES5 expression after 48 h, confirming inhibitor activity (n = 3 independent experiments).

(F) DAPT rapidly reduced Ki67⁺ cells after 48 h (n = 3 independent experiments).

(G) Example FACS-based cell-cycle analysis in Basal and DAPT cultures (n = 3 independent experiments).

(H) DAPT reduced the number of progenitors entering S phase within 24 h.

(I) Neurons generated after DAPT treatment were almost exclusively TBR1⁺ at D55 (n = 3 independent experiments).

(J) qRT-PCR confirmed that DAPT treatment reduced CTIP2, BRN2, CUX1, and SATB2 expression (n = 3 independent experiments).

(K) Schematic of DAPT-induced cell-cycle exit showing that D20 progenitors are competent to generate early-born, layer-VI TBR1⁺ neurons.

Data are mean ± SEM; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. Scale bars, 100 μm.