Figure 3.

WNT/CTNNB1 Signaling Regulates the Proliferation and Neurogenesis of Human MGE Progenitors

(A and B) Four-hour BrdU-incorporation analysis showed lower proliferation rates in KO-MGE progenitors at day 25. The KO-MGE progenitors also showed decreased Ki67 and PH3 labeling compared with that of the WT-MGE progenitors control (A). Scale bar, 50 μm. Statistical results are shown in (B). n = 236–1,027 cells in at least three independent experiments; unpaired t test, ^∗p < 0.05, ^∗∗∗p < 0.001.

(C and D) Immunostaining (C) and statistical analysis (D) demonstrated that KO-MGE progenitors increased cell populations stained with DCX at day 25. Scale bar, 50 μm. n = 258–557 cells in at least three independent experiments; unpaired t test, ^∗∗∗p < 0.001.

(E) WT- and KO-MGE progenitors differentiated from hESCs at day 25 were plated for an additional 30 days, and mRNA expression levels of TUJ1, GAD67, and SST were analyzed by qRT-PCR. At least three independent experiments were performed; unpaired t test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

(F) Construction of doxycycline (Dox)-inducible CTNNB1 S33Y rescue (RE) hESCs on the background of CTNNB1-KO was done through lentiviral vector infection. Western blotting confirmed induced expression of CTNNB1 after treatment with 0.1 μg/mL Dox for 5 days.

(G and H) Immunostaining (G) and statistical analysis (H) demonstrated that Dox treatment in RE-MGE progenitors at days 17–25 significantly increased cell populations with BrdU incorporation or Ki67 and PH3 immunolabeling. Scale bar, 50 μm. n = 85–333 cells in at least three independent experiments; unpaired t test, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

(I and J) Dox treatment in RE-MGE progenitors at days 17–25 significantly decreased cell populations immunolabeled with DCX (I). Scale bar, 50 μm. Statistical results were shown in (J). n = 130–264 cells in at least three independent experiments; unpaired t test, ^∗p < 0.05.

(K and L) CTNNB1 S33Y OE-hESCs were targeted to the MGE, with 0.1 μg/mL Dox addition from day 17 to 25. At day 25, Dox-treated MGE progenitors showed increased populations of cells labeled with BrdU, Ki67, and PH3 (K). Scale bar, 50 μm. Statistical results were shown in (L). n = 98–570 cells in at least three independent experiments; unpaired t test, ^∗p < 0.05, ^∗∗p < 0.01.

(M and N) Immunostaining (M) and statistical analysis (N) demonstrated that CTNNB1-OE decreased cell populations immunolabeled with DCX in MGE progenitors. Scale bar, 50 μm. n = 137–271 cells in at least three independent experiments; unpaired t test, ^∗p < 0.05.

(O) At day 25, the OE-MGE progenitors with or without Dox treatment expressed FOXG1, but not HOXB4. Scale bar, 50 μm.

(P) The OE-MGE progenitors with or without Dox treatment were immunolabeled by NKX2.1 at day 25. Scale bar, 50 μm.