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Understanding human embryonic ventral midbrain is of major interest for Parkinson's disease. However, the cell types, their gene expression dynamics, and their relationship to commonly used rodent models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types, including five subtypes of radial glia-like cells and four progenitors. In the mouse, two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species, but with clear differences in cell proliferation, developmental timing, and dopaminergic neuron development. Additionally, we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells, at a single-cell level. Thus, our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Midbrain dopaminergic (mDA) neuron development has been an intense area of research during recent years. This is due in part to a growing interest in regenerative medicine and the hope that treatment for diseases affecting mDA neurons, such as Parkinson's disease (PD), might be facilitated by a better understanding of how these neurons are specified, differentiated and maintained in vivo. This knowledge might help to instruct efforts to generate mDA neurons in vitro, which holds promise not only for cell replacement therapy, but also for disease modeling and drug discovery. In this Primer, we will focus on recent developments in understanding the molecular mechanisms that regulate the development of mDA neurons in vivo, and how they have been used to generate human mDA neurons in vitro from pluripotent stem cells or from somatic cells via direct reprogramming. Current challenges and future avenues in the development of a regenerative medicine for PD will be identified and discussed., (© 2015. Published by The Company of Biologists Ltd.)

Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1(-/-) mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1(-/-);Wnt5a(-/-) mice revealed a greater loss of Nurr1(+) cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson's disease.

Signaling mechanisms involving Wnt/beta-catenin and sonic hedgehog (Shh) are known to regulate the development of ventral midbrain (vMB) dopamine neurons. However, the interactions between these two mechanisms and how such interactions can be targeted to promote a maximal production of dopamine neurons are not fully understood. Here we show that conditional mouse mutants with region-specific activation of beta-catenin signaling in vMB using the Shh-Cre mice show a marked expansion of Sox2-, Ngn2-, and Otx2-positive progenitors but perturbs their cell cycle exit and reduces the generation of dopamine neurons. Furthermore, activation of beta-catenin in vMB also results in a progressive loss of Shh expression and Shh target genes. Such antagonistic effects between the activation of Wnt/beta-catenin and Shh can be recapitulated in vMB progenitors and in mouse embryonic stem cell cultures. Notwithstanding these antagonistic interactions, cell-type-specific activation of beta-catenin in the midline progenitors using the tyrosine hydroxylase-internal ribosomal entry site-Cre (Th-IRES-Cre) mice leads to increased dopaminergic neurogenesis. Together, these results indicate the presence of a delicate balance between Wnt/beta-catenin and Shh signaling mechanisms in the progression from progenitors to dopamine neurons. Persistent activation of beta-catenin in early progenitors perturbs their cell cycle progression and antagonizes Shh expression, whereas activation of beta-catenin in midline progenitors promotes the generation of dopamine neurons.