Interactions of Wnt/β-Catenin Signaling and Sonic Hedgehog Regulate the Neurogenesis of Ventral Midbrain Dopamine Neurons

Cell cycle progression of the DA progenitors in vMB of Shh–Cre;β-Ctn^Ex3/+ mutants. A–F, Stabilization of β-catenin in vMB results in increased proliferation in early progenitors (2 h BrdU injection scheme) in the ventricular zone at E12.5 (E, F) but not at E10.5 and E11.5 (A–D). Student's t test (n = 3); ns, Not significant. G, H, Neuronal birthdating experiments using a 24 h BrdU injection scheme also reveal a robust increase in BrdU⁺ cells in vMB. However, there is a reduction in newly born DA neurons, highlighted by BrdU and TH double-positive (BrdU⁺;TH⁺) cells in Shh–Cre;β-Ctn^Ex3/+ mutants (arrowheads in the insets). I, J, Similarly, the number of PH3⁺ progenitors also increase in Shh–Cre;β-Ctn^Ex3/+ mutants. Scale bars: (in D) A–D, 50 μm; (in J) E–J, 100 μm. K, L, Cell cycle exit experiments show that fewer progenitor cells exit cell cycle in Shh–Cre;β-Ctn^Ex3/+ mutants. Scale bar (in L): K, L, 50 μm. M–Q, Quantification results confirm the increases in BrdU⁺ progenitors (2 and 24 h injection schemes) (M, N), the reduced production of BrdU⁺;TH⁺ neurons (O), the increase in PH3⁺ progenitors (P), and the decrease in cell cycle exit (Q) after the activation of β-catenin in vMB.

Ventral midbrain progenitors in Shh–Cre;β-Ctn^Ex3/+ mutants show a significant reduction in DA neurons in vivo and in vitro. A–F, Compared with control (β-Ctn^Ex3/+), vMB of Shh–Cre;β-Ctn^Ex3/+ mutants show much fewer DA neurons at E12.5 (A, B) and E18.5 (C–F). Scale bars: (in B), A, B, 100 μm; (in F) C–F, 250 μm. IPN, Interpeduncular nucleus. G, Quantification by stereology confirms the reduced number of DA neurons at E12.5 and P0. Student's t test, n = 3. H–M, Progenitors derived from the vMB of control (β-Ctn^Ex3/+) and Shh–Cre;β-Ctn^Ex3/+ mutants can be differentiated into DA neurons during the addition of Shh (J, K) and Wnt5a (L, M). Scale bar (in M): H–M, 50 μm. N, However, the relative number of TH⁺ cells per areas observed in Shh–Cre;β-Ctn^Ex3/+ mutants are significantly less in the presence of Shh (left) but similar to control at higher concentration of Wnt5a (100 ng/ml) (right). Culture scheme is shown on the top of N. The numbers of TH⁺ neurons per area are normalized to control for each genotype. Student's t test (n = 3); ns, Not significant. DIV, days in vitro.

Activation of β-catenin in vMB antagonizes Shh expression and reduces Shh target genes. A, B, Shh mRNA expression is slightly reduced at E10.5 in vMB of Shh–Cre;β-Ctn^Ex3/+ mutants. Scale bar (in B): A, B, 250 μm. C–F, By E12.5, Shh mRNA (C, D) and protein (E, F) expression are severely reduced in the vMB of Shh–Cre;β-Ctn^Ex3/+ mutants. G, H, Two representative images show the extensive colocalization of Shh (green) and the radial glia marker Nestin (red) (G) and Shh and RC-2 (H). Scale bar: H, 100 μm. I, J, Shh downstream target gene cyclin D1 show a similar reduction in the vMB of Shh–Cre; β-Ctn^Ex3/+ mutants. Scale bar (in J): C–H, 250 μm. K–N, Compared with control (β-Ctn^Ex3/+), another Shh target Foxa2 shows no detectable reduction in Shh–Cre;β-Ctn^Ex3/+ mutants at E10.5 (K, L) but is significantly downregulated at E12.5 (M, N). Scale bars: L, 100 μm; N, 50 μm. O, P, In contrast, expression of Nkx6.1 and Nkx2.2 in vMB shows no detectable changes in Shh–Cre;β-Ctn^Ex3/+ mutants. Scale bar: N, 100 μm. Q, Quantification of Foxa2⁺ progenitors at E10.5 and E12.5. Student's t test (n = 3). ns, Not significant.

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).

Antagonistic effects between Wnt/β-catenin and Shh in generating DA neurons from mESCs. A, Schematic diagrams illustrating the culture conditions to generate DA neurons from mESCs. Mouse ESCs are cultured in the presence of SRM, followed by basal conditions, including SRM plus FGF8 (for 3 d), SRM plus FGF2 and FGF8 (for 3 d), and finally N2 supplement plus ascorbic acid (AA), GDNF, and BDNF (for 3 d). Modified treatment conditions include the addition of Shh, CT99021, or Shh and CT99021 from day 5 to 11. B, Treatment with Shh (200 ng/ml) or CT99021 (2 μm) alone increases the DA neuron numbers. C, Combined treatments with Shh and CT99021 do not show additive or synergistic effects in generating more DA neurons from mESCs. In contrast, higher concentration of Shh (1250 ng/ml) antagonizes the effects of CT99021 on DA neuron yield. D, Similarly, the GSK3β inhibitor CT99021 also antagonizes on the effect of Shh in the generation of DA neuron in a range of concentrations (n = 3). DIV, Days in vitro; CON, control.

Increased DA neurons in Th–IRES–Cre;β-Ctn^Ex3/+ mutants. A–H, Compared with control (β-Ctn^Ex3/+), the number of DA neurons is increased in Th–IRES–Cre;β-Ctn^Ex3/+ mice at E11.5 (A, B), E12.5 (C, D), P0 (E, F), and P21 (G, H). Scale bars: D, H, 200 μm. I, Quantification using stereology confirms the increased number of DA neurons at E11.5, E12.5, P0, and P21. Student's t test, n = 3. J–M, Th–IRES–Cre;β-Ctn^Ex3/+ mice show a persistent increase in the number of postmitotic DA precursors (Nurr1⁺;TH⁻) at E11.5 and E12.5. Scale bar: J–M, 50 μm. N, Quantitative analyses for the number postmitotic DA precursors (Nurr1⁺;TH⁻) at E11.5 and E12.5. O–R, Neuronal birthdating experiments reveal increases of newly generated DA neurons (BrdU⁺;TH⁺) from E10.5 to E11.5 and from E11.5 to E12.5. The arrowheads indicate there are more BrdU⁺;TH⁺ cells in the mutants. S, Quantification confirms the increased number of BrdU⁺;TH⁺ cells at E11.5 and E12.5.