Functional roles of Nurr1, Pitx3, and Lmx1a in neurogenesis and phenotype specification of dopamine neurons during in vitro differentiation of embryonic stem cells.

To elucidate detailed functional mechanisms of key fate-determining transcription factors (eg, Nurr1, Pitx3, and Lmx1a) and their functional interplay for midbrain dopamine (mDA) neurons, we developed highly efficient gain-of-function system by transducing the neural progenitors (NPs) derived from embryonic stem cells (ESCs) with retroviral vectors, allowing the analysis of downstream molecular and cellular effects. Overexpression of each factors, Nurr1, Pitx3, and Lmx1a robustly promoted the dopaminergic differentiation of ESC-NP cells exposed to sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8). In addition, each of these factors directly interacts with potential binding sites within the tyrosine hydroxylase (TH) gene and activated its promoter activity. Interestingly, however, overexpression of Nurr1, but not of Pitx3 or Lmx1a, generated a significant number of nonneuronal TH-positive cells. In line with this, Pitx3 and Lmx1a, but not Nurr1, induced expression of the Ngn2 gene, which is critical for neurogenesis. We also observed that Pitx3 directly bound to its potential binding sites within the Ngn2 gene and the pan-neuronal marker β-tubulin III gene, suggesting that Pitx3 contributes to mDA neurogenesis by directly regulating these genes. Taken together, our data demonstrate that key mDA regulators (Nurr1, Pitx3, and Lmx1a) play overlapping as well as distinct roles during neurogenesis and neurotransmitter phenotype determination of mDA neurons.