Recent developments in molecular programming of mesodiencephalic dopaminergic (mdDA) neurons have led to the identification of many transcription factors playing a role in mdDA specification. LIM homeodomain transcription factor Lmx1a is essential for chick mdDA development, and for the efficient differentiation of ES-cells towards a dopaminergic phenotype. In this study, we aimed towards a more detailed understanding of the subtle phenotype in Lmx1a-deficient (dreher) mice, by means of gene expression profiling. Transcriptome analysis was performed, to elucidate the exact molecular programming underlying the neuronal deficits after loss of Lmx1a. Subsequent expression analysis on brain sections, confirmed that Nurr1 is regulated by Lmx1a, and additional downstream targets were identified, like Pou4f1, Pbx1, Pitx2, C130021l20Rik, Calb2 and Rspo2. In line with a specific, rostral-lateral (prosomer 2/3) loss of expression of most of these genes during development, Nurr1 and C130021l20Rik were affected in the SNc of the mature mdDA system. Interestingly, this deficit was marked by the complete loss of the Wnt/b-catenin signaling activator Rspo2 in this domain. Subsequent analysis of Rspo2−/− embryos revealed affected mdDA neurons, partially phenocopying the Lmx1a mutant. To conclude, our study revealed that Lmx1a is essential for a rostral-lateral subset of the mdDA neuronal field, where it might serve a critical function in modulating proliferation and differentiation of mdDA progenitors through the regulation of the Wnt activator Rspo2.

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 2 (PRC2). 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 (NPCs); 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 (DA) 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 (cKOs) 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.

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.

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.

During development, mesodiencephalic dopaminergic (mdDA) neurons form into different molecular subsets. Knowledge of which factors contribute to the specification of these subsets is currently insufficient. In this study, we examined the role of Tcf4, a member of the E-box protein family, in mdDA neuronal development and subset specification. We show that Tcf4 is expressed throughout development, but is no longer detected in adult midbrain. Deletion of Tcf4 results in an initial increase in TH-expressing neurons at E11.5, but this normalizes at later embryonic stages. However, the caudal subset marker Nxph3 and rostral subset marker Ahd2 are affected at E14.5, indicating that Tcf4 is involved in correct differentiation of mdDA neuronal subsets. At P0, expression of these markers partially recovers, whereas expression of Th transcript and TH protein appears to be affected in lateral parts of the mdDA neuronal population. The initial increase in TH-expressing cells and delay in subset specification could be due to the increase in expression of the bHLH factor Ascl1, known for its role in mdDA neuronal differentiation, upon loss of Tcf4. Taken together, our data identified a minor role for Tcf4 in mdDA neuronal development and subset specification.