**2. The Wnt/β-catenin signaling pathway**

The Wnt signaling pathway, conserved from low animals to primates, was originally identi‐ fied as the morphogenic signaling for organogenesis. Among three branches of Wnt signal‐

© 2013 Chen; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Chen; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ing pathways, the best studied one is the canonical Wnt pathway, which is highlighted by βcatenin-dependent regulation of down-streaming genes. The canonical Wnt ligands, e.g. Wnt1, Wnt2 and Wnt3a, can be secreted by surrounding neuronal and glial cells in the nerv‐ ous system, bind to Frizzled and Lrp5/6 receptors in the target cells. At absence of binding with ligand Wnts, a protein complex in the cytoplasm, namely Axin2/GSK3β/APC complex makes phosphorylation of β-catenin and degradation of phosphorylated β-catenin, and keeps cytoplasmic β-catenin at such a low level that β-catenin can not be translocated into nucleus. Upon Wnt stimulation, the Axin2/GSK3β/APC complex can be deaggregated, the cytoplasmic β-catenin is accumulated and increased β-catenin imported into nucleus. In the nucleus, β-catenin is recruited by transcription factors TCF1-4 to the promoter regions of the target genes for specific biological effects. These TCF-4 downstream targeting genes include c-myc, mmp-7, cyclin D1, CD44 that are actively and mainly involved in cell proliferation, cycling and cell differentiation (Figure 1). On the other hand, the non-canonical Wnt signal‐ ing pathways, i.e. PCP pathway and Ca2+ pathway, Wnt ligands bind to Frizzled receptors, then activate GTPase or increase intracellular Ca2+, transmitting signals by JNK cascade or without any nucleus events [4-6].

**3. Role of Wnt/β-catenin signaling in facilitating DA neuronal**

The canonical Wnt/β-catenin signaling pathways is critical for generation of DA neurons during development. Differential regulation of midbrain DA neurogenesis by Wnt1, Wnt3a, and Wnt5a was well studied [9]. The β-catenin was detected in DA precursor cells and β-catenin signaling took place in the precursor cells by assessment of TOPgal reporter mice. Wnt3a promoted prolif‐ eration of precursor cells expressing the orphan nuclear receptor-related factor 1 (Nurr1) but did not increase the number of DA neurons. The Wnt1 and Wnt5a increased the number of midbrain DA neurons in E14.5 possibly by two mechanisms. Wnt1 predominantly increased the prolifera‐ tion of Nurr1+ precursors that acquired a neuronal DA phenotype, up-regulated cyclins D1 and D3, and down-regulated p27 and p57 expression. In contrast, Wnt5a increased proportion of Nurr1+ precursors and up-regulated expression of Ptx3 and c-ret mRNA. Moreover, the soluble cysteine-rich domain of Frizzled-8 (a Wnt inhibitor) blocked endogenous Wnts and effects of Wnt1 and Wnt5a on proliferation and acquisition of DA phenotype. For the embryonic expres‐ sion, Wnt1 was throughout of midbrain at E8.5, and then restricted to the roof plate, a subset of floor plate cells and isthemus of midbrain at E9.5. From E10 to E12, Wnt2 was observed in the ven‐ tral midbrain, with highest in the intermediate and marginal zone of ventral midbrain. Wnt3a was expressed in dorsal midbrain of rat at E11.5. The Wnt5a appeared at E9.5 and became restrict‐ ed to the floor plate of midbrain from E11.5 to E13.5. Functionally, mutation of Wnt1 led to re‐ duced DA neurons in late embryos. Mechanistic study showed that Wnt1 and its downstream gene Lmx1 formed a loop to regulate the expression of Octx2, Nurr1 and Pitx3, thereby establish‐ ing identity of DA precursors *in vivo* [10]. This Wnt1-lmx1a regulatory loop synergistically con‐ trolled DA differentiation in the midbrain by antagonizing Shh signaling pathway [11]. Wnt 2 mutation resulted in a decrease in proliferation of DA progenitors and subsequently loss of DA neurons, partially by phosphorylation of Lrp5/6 and Dishevelled 2/3. Wnt 3a promoted the pro‐ liferation of Nurr1-positive DA progenitors. The Wnt5a, derived by the astrocytes and radial glial cells, was demonstrated to promote cell fate commitment of precursors into DA neurons and development of A9-A10 DA neurons *in vivo* [12, 13]. The Wnt5a also regulated DA axon growth and guidance in midbrain development [14]. In mouse embryo at E11.5, Wnt5a was abundantly expressed in the ventral midbrain where it promoted DA neurite and axonal growth. By E14.5, when DA axons were approaching their striatal target, Wnt5a caused DA neurite re‐ traction. Co-culture of ventral midbrain explants with Wnt5a-overexpressing cell aggregates re‐ vealed that Wnt5a was capable of repelling DA neurites. Antagonism experiments revealed that the effects of Wnt5a were mediated by the Frizzled receptors and by small GTPase, Rac1. More‐ over, this effect was specifically blocked by Wnt5a antibody. Role of Wnt5a in DA neuronal axon morphogenesis was further verified in Wnt5a-/-mice, where fasciculation of the medial fore‐ brain bundle as well as the density of DA neurites and striatal terminals were disrupted. Al‐ though Wnts can function via intracellularβ-catenin, Ca2+, and JNK signaling, the canonical Wnt/β-catenin signaling pathway shows a major position in regulation of DA neurogenesis. It appeared that all Wnt1, Wnt3a and Wnt5a act in proliferation and DA differentiation of precur‐ sor cells with sequence of proliferation stimulating effect of Wnt1≥Wnt3a≥Wnt5a, or differentia‐ tion facilitating effects of Wnt5a≥Wnt1≥Wnt3a. These findings have evidenced that the Wnt/β-

Roles of Wnt/β-Catenin Signaling in Controlling the Dopaminergic Neuronal Cell Commitment of Midbrain and

Therapeutic Application for Parkinson's Disease

http://dx.doi.org/10.5772/53282

143

**development**

**Figure 1.** The canonical Wnt/β-catenin signaling pathway in regulation of downstream target genes of DA neurogen‐ esis (*From Ref. 48, Ding, et al., 2011*)
