Figure 5. Ventral midbrain progenitor cultures show antagonistic effect between canonical Wnt/β-catenin signaling and Shh. A, Schematic diagrams illustrating the two culture conditions for DA progenitors. In the “single or combined treatment” paradigm, vMB progenitors are treated with Shh, Wnt1, the GSK3β inhibitor CT99021 alone, or with a combination of Shh and Wnt1, or Shh and CT99021 (B–L). In the “sequential treatment” paradigm, the vMB progenitors are treated with Shh or CT99021 from day 1 to 3 and then switched to CT99021 or Shh from day 3 to 5 (M, N). B–J, Compared with control (B, E), treatments with Shh or Wnt1 alone increase the number of DA neurons in a dose-dependent manner, with the maximal effect at 250 ng/ml (C, F, H, I). Similarly, treatments with the GSK3β inhibitor CT99021 can also increase the number of DA neurons (J). K, L, To determine whether simultaneous treatments of Wnt1 and Shh can maximize the production of DA neurons, we treat the progenitor cultures with optimal concentration of Shh (250 ng/ml) and increasing amounts of Wnt1 or CT99021 (D, G, K, L). Combined treatment of Shh and Wnt1 show a very modest increase in DA neuron number at 50 and 250 ng/ml Wnt1 but not at high dose (1250 ng/ml). On the contrary, combined treatments of Shh and CT99021 show an antagonistic effect (n = 4). Scale bar (in G): B–G, 50 μm. M, N, Compared with single treatment with CT99021 or Shh, sequential treatment of CT99021 and Shh decreases the DA neuron number (n = 3).