Wnt2 Regulates Progenitor Proliferation in the Developing Ventral Midbrain

Abstract

Wnts are secreted, lipidated proteins that regulate multiple aspects of brain development, including dopaminergic neuron development. In this study, we perform the first purification and signaling analysis of Wnt2 and define the function of Wnt2 in ventral midbrain precursor cultures, as well as in Wnt2-null mice in vivo. We found that purified Wnt2 induces the phosphorylation of both Lrp5/6 and Dvl-2/3, and activates ␤-catenin in SN4741 dopaminergic cells. Moreover, purified Wnt2 increases progenitor proliferation, and the number of dopaminergic neurons in ventral midbrain precursor cultures. In agreement with these findings, analysis of the ventral midbrain of developing Wnt2-null mice revealed a decrease in progenitor proliferation and neurogenesis that lead to a decrease in the number of postmitotic precursors and dopaminergic neurons. Collectively, our observations identify Wnt2 as a novel regulator of dopaminergic progenitors and dopaminergic neuron development.

Wnt5a cooperates with canonical Wnts to generate midbrain dopaminergic neurons in vivo and in stem cells

“…Recent reports have indicated that Wnt/ β-catenin signaling is required for midbrain DA neurogenesis (30,31), but it is not known which of the multiple canonical Wnts expressed in the VM (13)(14)(15) is/are required for DA neurogenesis. In our study we focused on Wnt1 because Wnt1 −/− mice, unlike Wnt2 −/− mice, for instance (16), show a strong sequential midbrain and DA neuron phenotype (18)(19)(20)(21)(22). Because DA neurons are born in the midbrain FP, we first examined the expression of the FP and basal plate (BP) markers, Shh and the Shh-target gene, Foxa2, both of which are required for DA neuron development (31,(33)(34)(35).…”

“…These include Wnt1, Wnt2, and Wnt3a, which activate the Wnt/β-catenin pathway (14,16), and Wnt5a, which activates the Wnt/Rac1 pathway in dopaminergic (DA) cells (17). Wnt1-knockout mice show a partial segmental deletion of the midbrain and hindbrain regions (18,19) resulting from multiple sequential defects, including altered Otx2 and Pitx3 expression, reduced progenitor proliferation, and death of midbrain DA neurons (18)(19)(20)(21)(22).…”

Canonical and noncanonical Wnt signaling in neural stem/progenitor cells

“…Wnt-1 KO Altered central and peripheral neuronal development during initial axonogenesis [54] Wnt-1 KO Impaired midbrain development [55] Wnt-1 dominant negative Impaired hippocampal neurogenesis and spatial and object recognition memory [56,57] Wnt-1 overexpression Reduced neural differentiation of mESCs (also by treatment with lithium chloride) [58] Wnt-1 KO Increased differentiation into DA neurons in KO mESCs [59] Wnt-2 KO Decreased progenitor proliferation and neurogenesis in the ventral midbrain [60] Wnt-2 overexpression Induced dendritic arborization in hippocampal progenitors [61] Wnt-3 overexpression Increased differentiation of cortical intermediate [43] progenitors Wnt-3 overexpression Induced differentiation through cleavage of RYK in cortical progenitors [62] Wnt-3a KO Loss of the hippocampus [42] Recombinant Wnt-3a Induced GABAergic neuronal differentiation through RYK, reduced oligodendrogenesis [63] Recombinant Wnt-3a Induced differentiation of hESCs [64] Recombinant/purified Wnt-3a…”

International Union of Basic and Clinical Pharmacology. LXXX. The Class Frizzled Receptors

“…The next step is immobilized metal affinity chromatography followed by Superdex gel filtration and, finally, heparin cation exchange (affinity) chromatography (Willert, 2008). This protocol with minor modifications has successfully been used to purify WNT-2, WNT-3A, -5A, -7A, -16, D. melanogaster WNTD, and Wingless (Willert, 2008;Sousa et al, 2010). Even though several WNTs could successfully be purified according to the protocol establish by Karl Willert, it should be emphasized that remaining WNTs still resist purification in a biological active form.…”

“…Purification of WNT-2, WNT-3A, and WNT-5A from conditioned medium was described by the Willert group and others (Willert et al, 2003;Schulte et al, 2005;Mikels and Nusse, 2006;Willert, 2008;Sousa et al, 2010). For this purpose, conditioned medium from WNT overexpressing mammalian cells is harvested and fractionated with affinity chromatography using BlueSepharose, which binds rather selectively to WNT proteins.…”