Dickkopf 3 Promotes the Differentiation of Substantia Nigra Dopaminergic Neurons In Vivo and from Pluripotent Stem Cells In Vitro

WNT1/beta-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons including the Substantia nigra pars compacta (SNc) subpopulation, whose degeneration is a hallmark of Parkinson's Disease (PD). However, the precise functions of WNT/beta-catenin signaling in this context remain unknown. Using mutant mice, primary ventral midbrain (VM) cells and pluripotent stem cells (mouse embryonic stem cells and induced pluripotent stem cells), we show that Dickkopf 3 (DKK3), a secreted glycoprotein that modulates WNT/beta-catenin signaling, is specifically required for the correct differentiation of a rostrolateral mdDA precursor subset into SNc DA neurons. Dkk3 transcription in the murine VM coincides with the onset of mdDA neurogenesis and is required for the maintenance of LMX1A and consequently PITX3 expression in rostrolateral mdDA precursors, without affecting the proliferation or specification of their progenitors. Treatment of primary VM cells or differentiating pluripotent stem cells with recombinant WNT1 and/or DKK3 proteins consistently increases the proportion of mdDA cells with SNc DA neuron identity and promotes their survival in vitro. The SNc DA pro-differentiation and pro-survival properties of DKK3, together with its known anti-tumorigenic effect, therefore make it an ideal candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD. We performed gene expression microarray analysis on iPSC-derived and FACS-sorted GFP-positive Pitx3GFP/+ mdDA neurons, differentiated in the presence or absence of recombinant human WNT1 and recombinant human DKK3. In addition, we analysed primary and FACS-sorted GFP-positive Pitx3+/GFP mdDA neurons isolated from the E13.5 and E14.5 ventral midbrain of Pitx3+/GFP embryosWNT1/β-连环蛋白（beta-catenin）信号通路在中脑多巴胺能（mesodiencephalic dopaminergic, mdDA）神经元——包括黑质致密部（Substantia nigra pars compacta, SNc）亚群——的生成过程中发挥关键作用，而该亚群神经元的变性是帕金森病（Parkinson's Disease, PD）的标志性病理特征。然而，WNT/β-连环蛋白信号通路在此背景下的精确功能仍未明确。本研究借助突变型小鼠、原代腹侧中脑（ventral midbrain, VM）细胞以及多能干细胞（含小鼠胚胎干细胞与诱导多能干细胞）开展实验，证实调控WNT/β-连环蛋白信号通路的分泌型糖蛋白Dickkopf 3（DKK3），是吻侧外侧mdDA前体亚群定向分化为SNc型多巴胺能神经元的特异性必需因子。小鼠VM组织中的Dkk3转录水平与mdDA神经发生的起始阶段相吻合，且该基因对维持吻侧外侧mdDA前体中LMX1A的表达至关重要，进而保障PITX3的正常表达，却不会影响其前体细胞的增殖与定向特化。向原代VM细胞或正在分化的多能干细胞施加重组WNT1和/或DKK3蛋白，可持续稳定地提升具有SNc型多巴胺能神经元表型的mdDA细胞比例，并在体外促进这些细胞的存活。鉴于DKK3兼具促SNc型多巴胺能神经元分化与存活的特性，加之其已知的抗肿瘤作用，该蛋白成为优化帕金森病再生与神经保护治疗策略的理想候选分子。本研究对两类样本开展了基因表达微阵列分析：其一为经重组人源WNT1与重组人源DKK3处理或未处理的、由诱导多能干细胞衍生且经荧光激活细胞分选（fluorescence-activated cell sorting, FACS）得到的GFP阳性Pitx3GFP/+ mdDA神经元；其二为从Pitx3+/GFP胚胎的胚胎发育第13.5天（E13.5）与第14.5天（E14.5）腹侧中脑分离得到的、经FACS分选的GFP阳性Pitx3+/GFP mdDA神经元。