Interplay between Wnt, Nurr1 and En1 signaling in vitro and in vivo. (A) Model adapted from Kitagawa et al. : Wnt signaling via β-catenin enhances the transcriptional activity of Nurr1 in cells at Nurr1 responsive elements (NREs). In the absence of β-catenin, Nurr1 associates with T cell factor/lymphoid enhancer factor (TCF/LEF) in co-repressor complexes on NREs. After activation of Wnt signaling, β-catenin interacts with Nurr1 on NREs, competing with TCF/LEF for Nurr1 binding, resulting in the disruption of the co-repressors from the Nurr1 complex and the concomitant recruitment of coactivators. (B) Model adapted from Kitagawa et al. : on the other hand, Nurr1 was observed to slightly modulate, in a negative way, the canonical Wnt signaling through association with the TCF/LEF region. After Wnt stimulation, β-catenin competed with Nurr1 for Lef binding on the TCF/LEF promoter site and disrupted Nurr1 binding, promoting Wnt-target gene transcription. (C) Several studies in Drosophila and chick embryos have described interactions between En1/engrailed (en) and the Wnt/wg signaling pathway whereby engrailed expression is dependent on Wnt/wg signaling and vice versa. However, in Drosophila, engrailed expressing cells did not have active wg signaling. From mice studies it is known that Wnt signaling regulates En1 expression early in midbrain development. Whether the reverse happens in the mouse midbrain is not known. (D) In one cell culture study , it was observed that En1 can function as a negative regulator of β-catenin transcriptional activity in a post-translational manner (that is, by affecting β-catenin protein levels only). (E) Three questions remain currently unsolved: first, whether En1 cooperates with Nurr1 during mdDA development; second, whether Nurr1, En1 and canonical Wnt signaling cooperate in later stages of mdDA neuron development, such as in mdDA neuron specification; and third, whether Nurr1 and/or En1 regulate canonical Wnt signaling during mdDA neuron development.