1. Interactions of Wnt/β-Catenin Signaling and Sonic Hedgehog Regulate the Neurogenesis of Ventral Midbrain Dopamine Neurons
Maneeshi Prasad
Wnt/β-Catenin Signaling Seminar class Spring 2011
April 29th 2011

2. Wnt/β-catenin signaling

3. Wnt/β-catenin signaling in vMB
Wnts regulate DA neuron development in vMB by regulating:
- Proliferation, specification, neurogenesis of
DA progenitors in vMB
- Wnt also regulates survival of DA neurons
β-catenin maintains adherent junctions and cell cycle progression in DA progenitors in vMB

4. Targets of Wnt/β-catenin signaling in vMB
Otx2 (Orthodenticle homeobox 2)
Otx2 is required from E10.5 onward to regulate neuronal subtype identity and neurogenesis in the midbrain (PMID: )
Limx1a (LIM homeobox)
Involved in development of DA neurons
Allelic variations in three LMX1A single nucleotide polymorphisms (SNPs) were related to risk of Parkinson's disease (PMID: )
Both genes form autoregulatory loop with Wnt 1 gene

5. Shh Signaling

7. Sonic Hedgehog (Shh) signaling
Shh and FoxA2 autoregulatory loop is involved in DA neuron development
Foxa1 and Foxa2 are required throughout multiple phases of DA neuron development
- Expression of Nurr1 and En1 in immature neurons
- TH and AADC in mature neurons
- Higher dose for differentiation and lower for progenitor specification
Aging foxa2+/− mice spontaneously develop Parkinsonism (Kittappa et al., 2007)

8. Interaction between Wnt/β-Catenin and Sonic Hedgehog Signaling

9. Mice strains 1. β-catenin Exon3 mice (β-CtnEx3) [Wild type] 2. Shh–Cre
3. Tyrosine hydroxylase-internal ribosomal entry site-Cre (Th-IRES-Cre)
β-catenin Exon3 mice (-CtnEx3) crossed with Shh-Cre and Th-IRES-Cre to get:
A. Shh–Cre; β-CtnEx3/+
B. Th–IRES–Cre; β-CtnEx3/+

10. Shh–Cre; β-CtnEx3/+ mice
Cre recombinase is expressed under the control of Shh promoter in a tissue specific manner
Cre floxes Exon3 of β-catenin
Leads to loss of GSK3β phosphorylation site in Exon3 of β-catenin
High level of β-catenin activity in tissues expressing Shh

11. Schematics of inducible transgenic mice generation
β-catenin EX3 2
Flox 1 3
Cre
Shh-Cre
Or TH-Cre 1 3
Flox
β-catenin EX3/-

12. Experiments With transgenic inducible mice
Shh–Cre; β-CtnEx3/+
Th–IRES–Cre; β-CtnEx3/+
Primary culture from vMB of above mice strains
mES cell ceultures

13. Shh-Cre;β-CtnEx3/+ mutant mice show defects in limb development

14. Stabilization of β-catenin in vMB leads to expansion of DA progenitors.
Stabilization of β-catenin in vMB leads to expansion of DA progenitors. A, B, β-Catenin staining in the vMB of β-CtnEx3/+ and Shh–Cre;β-CtnEx3/+ mice at E12.5. Scale bar (in B), 50 μm. C, D, Immunofluorescent staining at E12.5 shows significant increases in the numbers of Sox2+, Ngn2+, and Sox2+;Ngn2+ progenitor at E12.5. E–N, In addition, the number of Otx2+ (E, F), Lmx1a+ (H, I), Lmx1b+ (J, K), and Nurr1+ (L, M) progenitors are all increased in Shh–Cre;β-CtnEx3/+ mutants. Scale bars: D, 40 μm; I, 50 μm; K, 100 μm; M, 50 μm. N, Quantifications of Lmx1a+, Otx2+, and Nurr1+ cells show a consistent increase of these progenitors in Shh–Cre;β-CtnEx3/+ mutants. The data are represented as number of positive cells per section as described previously (Tang et al., 2009). DAPI, 4′,6′-Diamidino-2-phenylindole.
©2010 by Society for Neuroscience

15. Cell cycle progression of the DA progenitors in vMB of Shh–Cre;β-CtnEx3/+ mutants.
Cell cycle progression of the DA progenitors in vMB of Shh–Cre;β-CtnEx3/+ 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-CtnEx3/+ mutants (arrowheads in the insets). I, J, Similarly, the number of PH3+ progenitors also increase in Shh–Cre;β-CtnEx3/+ 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-CtnEx3/+ 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.
©2010 by Society for Neuroscience

16. Stabilization of β-catenin in ventral midbrain does not increase cell death

17. Wnt increases DA progenitor by decreasing the cell cycle exit

18. Ventral midbrain progenitors in Shh–Cre;β-CtnEx3/+ mutants show a significant reduction in DA neurons in vivo and in vitro
Ventral midbrain progenitors in Shh–Cre;β-CtnEx3/+ mutants show a significant reduction in DA neurons in vivo and in vitro. A–F, Compared with control (β-CtnEx3/+), vMB of Shh–Cre;β-CtnEx3/+ 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-CtnEx3/+) and Shh–Cre;β-CtnEx3/+ 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-CtnEx3/+ 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.

19. Activation of β-catenin in vMB antagonizes Shh expression and reduces Shh target genes
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-CtnEx3/+ 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-CtnEx3/+ 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-CtnEx3/+ mutants. Scale bar (in J): C–H, 250 μm. K–N, Compared with control (β-CtnEx3/+), another Shh target Foxa2 shows no detectable reduction in Shh–Cre;β-CtnEx3/+ 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-CtnEx3/+ 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.

20. Persistent activation of Wnt/β-catenin pathway antagonizes Shh signaling and alters the neurogenic niche of DA neurons

21. Ventral midbrain progenitor cultures show antagonistic effect between canonical Wnt/β-catenin signalling and Shh
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).

22. Antagonistic effects between Wnt/β-catenin and Shh in generating DA neurons from mESCs
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.

23. Wnt and Shh do not have synergistic effect on DA neuron generation

24. Th–IRES–Cre; β-CtnEx3/+
Cre recombinase is expressed under the control of TH promoter in a tissue specific manner
Cre floxes Exon3 of β-catenin
Leads to loss of GSK3β phosphorylation site in Exon3 of β-catenin
High level of β-catenin activity in tissues expressing TH

25. Action of Tyrosine hydroxylase
TH gene
L-tyrosine
Tyrosine hydroxylase +
Tetrahydrobiopterin
(coenzyme)
Dihydroxyphenylalanine (DOPA)
AADC
Dopamine
DbH
Norepinephrine (noradrenaline)
PNMT
Epinephrine (adrenaline)

26. Increased DA neurons in Th–IRES–Cre;β-CtnEx3/+ mutants
Increased DA neurons in Th–IRES–Cre;β-CtnEx3/+ mutants. A–H, Compared with control (β-CtnEx3/+), the number of DA neurons is increased in Th–IRES–Cre;β-CtnEx3/+ 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-CtnEx3/+ 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.

27. Increase in newly born postmitotic DA precursors in
Th–IRES–Cre;β-CtnEx3/+ mutants
Increases in newly born postmitotic DA precursors in Th–IRES–Cre;β-CtnEx3/+ mutants. A–D, Neuronal birthdating experiments reveal increase newly generated postmitotic DA cells (BrdU+;Nurr1+) from E10.5 to E11.5 and from E11.5 to E12.5. Scale bar: A–D, 50 μm. E, Quantification confirms the increased number of BrdU+;Nurr1+ cells at E11.5 and E12.5. F, A proposed model to indicate that Wnt/β-catenin and Shh control the expression of distinct sets of transcription factors in the progenitors in vMB. Constitutive activation of Wnt/β-catenin suppresses the expression of Shh and its downstream targets, including cyclin D1 and Foxa2.

28. Cell type specific activation of Wnt/β-Catenin signaling in a subpopulation of midline progenitors increases neurogenesis and DA neurons

29. Conclusion/Discussion
Wnt/β-Catenin signaling promotes expansion of DA progenitors antagonizes Shh signaling and leads to reduced number of DA neurons
Cell-type specific activation of Wnt/β-Catenin signaling in midline progenitors leads to increased number of DA neurons
Broader Wnt/β-Catenin signaling activation promotes expansion of DA progenitors and restricted activation leads to more DA neurons

30. Thank You !!!!!!!!!