Over the last decade several components have been identified to be differentially expressed in subsets of mesodiencephalic dopaminergic (mdDA) neurons. These differences in molecular profile have been implied to be involved in the selective degeneration of the SNc neurons in Parkinson's disease. The emergence and maintenance of individual subsets is dependent on different transcriptional programs already present during development. In addition to the influence of transcription factors, recent studies have led to the hypothesis that modifications of histones might also influence the developmental program of neurons. In this study we focus on the histone methyltransferase EZH2 and its role in the development and maintenance of mdDA neurons. We generated two different conditional knock out (cKO) mice; an En1Cre driven cKO, for deletion of Ezh2 in mdDA progenitors and a Pitx3Cre driven cKO, to study the effect of post-mitotic deletion of Ezh2 on mdDA neurons maturation and neuronal survival. During development Ezh2 was found to be important for the generation of the proper amount of TH+ neurons. The loss of neurons primarily affected a rostrolateral population, which is also reflected in the analysis of the subset marks, Ahd2 and Cck. In contrast to early genetic ablation, post-mitotic deletion of Ezh2 did not lead to major developmental defects at E14.5. However, in 6 months old animals Cck was found ectopically in the rostral domain of mdDA neurons and Ahd2 expression was reduced in more mediocaudal positioned cells. In addition, Pitx3Cre driven deletion of Ezh2 led to a progressive loss of TH+ cells in the VTA and these animals display reduced climbing behavior. Together, our data demonstrates that Ezh2 is important for the generation of mdDA neurons during development and that during adult stages Ezh2 is important for the preservation of proper neuronal subset identity and survival.

The polycomb group proteins (PcGs) are a group of epigenetic factors associated with gene silencing. They are found in several families of multiprotein complexes, including Polycomb Repressive Complex (PRC) 2. EZH2, EED and SUZ12 form the core components of the PRC2 complex, which is responsible for the mono, di-and trimethylation of lysine 27 of histone 3 (H3K27Me3), the chromatin mark associated with gene silencing. Loss-of-function studies of Ezh2, the catalytic subunit of PRC2, have shown that PRC2 plays a role in regulating developmental transitions of neuronal progenitor cells; from self-renewal to differentiation and the neurogenic-to-gliogenic fate switch. To further address the function of EZH2 and H3K27me3 during neuronal development we generated a conditional mutant in which Ezh2 was removed in the mammalian isthmic (mid-hindbrain) region from E10.5 onward. Loss of Ezh2 changed the molecular coding of the anterior ventral hindbrain leading to a fate switch and the appearance of ectopic dopaminergic neurons. The correct specification of the isthmic region is dependent on the signaling factors produced by the Isthmic organizer (IsO), located at the border of the mid-and hindbrain. We propose that the change of cellular fate is a result of the presence of Otx2 in the hindbrain of Ezh2 conditional knock-outs and a dysfunctional IsO, as represented by the loss of Fgf8 and Wnt1. Our work implies that next to controlling developmental transitions, EZH2 mediated gene silencing is important for specification of the isthmic region by influencing IsO functioning and repressing Otx2 in the hindbrain. Kouwenhoven et al., 2016;Martinez et al., 1999). In this study we show that the En1Cre driven deletion of Ezh2 and a consequential loss of H3K27me3 leads to a disorganized IsO and ectopic expression of the transcription factor Otx2 in the hindbrain. In accordance with other studies in which Otx2 expression is expanded caudally, DA cells are formed in rhombomere (R) 1 at the expense of R1 born neuronal subtypes, including 5-HT neurons (Brodski et al., 2003;Kouwenhoven et al., 2016;Sherf et al., 2015). Together, our data suggest that next to transcription factors, gene repression via EZH2 and H3K27me3 are required for the repression of Otx2 in the hindbrain, maintenance of the IsO and the correct fate-determination of the isthmic area.