49 research outputs found

    EZH2 Influences mdDA Neuronal Differentiation, Maintenance and Survival

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    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

    miR-34b/c Regulates Wnt1 and Enhances Mesencephalic Dopaminergic Neuron Differentiation

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    The differentiation of dopaminergic neurons requires concerted action of morphogens and transcription factors acting in a precise and well-defined time window. Very little is known about the potential role of microRNA in these events. By performing a microRNA-mRNA paired microarray screening, we identified miR-34b/c among the most upregulated microRNAs during dopaminergic differentiation. Interestingly, miR-34b/c modulates Wnt1 expression, promotes cell cycle exit, and induces dopaminergic differentiation. When combined with transcription factors ASCL1 and NURR1, miR-34b/c doubled the yield of transdifferentiated fibroblasts into dopaminergic neurons. Induced dopaminergic (iDA) cells synthesize dopamine and show spontaneous electrical activity, reversibly blocked by tetrodotoxin, consistent with the electrophysiological properties featured by brain dopaminergic neurons. Our findings point to a role for miR-34b/c in neuronal commitment and highlight the potential of exploiting its synergy with key transcription factors in enhancing in vitro generation of dopaminergic neurons.Peer reviewe

    Expression of <i>Gli1</i>-eGFP and TH does not co-localize in the midbrain in the <i>Gli1</i>-eGFP-transgenic mouse line.

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    <p>(<b>A</b>) Model of the BAC-transgenic mouse line. This BAC-transgene contains several (minimum of 5) copies of eGFP which is transcribed under control of the <i>Gli1</i> promoter and immediately upstream of the <i>Gli1</i> sequence. When GLI1 expression is initiated by SHH-signaling, these cells start expressing eGFP. eGFP persists in the cell long after GLI1 has been broken down. Cells that expressed GLI1 can therefore be traced up to 2 days after expression of GLI1 has stopped. (<b>B</b>) Expression of <i>Gli1</i>-eGFP and TH does not co-localize at E11.5-E13.5 in the BAC-transgenic mouse-line. A strong separation between cells expressing <i>Gli1</i>-eGFP and TH<sup>+</sup>cells can be detected at all stages, but is most apparent at E11.5 and E12.5 (<b>1–4</b>). At E13.5 a few <i>Gli1</i>-eGFP expressing cells can be detected in the TH<sup>+</sup> area. However, no co-localization can be detected (<b>5–6</b>). A: Anterior; P: Posterior.</p

    Analysis of TH and GFP expression in multiple En1-/Pitx3-mutants at E14.5.

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    <p>(A) Immunohistochemistry of GFP and TH in different sections from medial to lateral encompassing the mdDA neuronal pool in Pitx3GPF/+ animals. (B-D) Same setup as described for A for (B) En1WT;<i>Pitx3</i>GFP/GFP, (C) En1Het;<i>Pitx3</i>GFP/GFP, and (D) En1KO;<i>Pitx3</i>GFP/GFP animals (matching sections with A). (D) Asterisk indicate the loss of TH immunoreactivity in more medial sections. Arrowheads indicate the presence of ectopic mdDA neurons.</p

    Quantitative PCR analysis of mdDA neurons in double En1KO;Pitx3GFP/GFP animals at E14.5.

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    <p>(A) Quantitative PCR demonstrates no further loss of <i>Ahd2</i> expression in En1KO;Pitx3GFP/GFP animals compared to En1WT;Pitx3GFP/GFP (P>0.05, n = 3/4). Significant loss of <i>Cck</i> expression in both the En1Het;Pitx3GFP/GFP (P<0.05, n = 4) and En1KO;Pitx3GFP/GFP animals (P<0.01, n = 3/4), compared to En1WT;Pitx3GFP/GFP. (B) Quantitative PCR demonstrates no changes in <i>Pbx1</i>, <i>Tle3</i>, <i>Tle4</i> and <i>Otx2</i> expression in En1KO;Pitx3GFP/GFP animals compared to En1WT;Pitx3GFP/GFP (P>0.05, n = 3/4). Significantly increased expression of <i>Pbx3</i> in En1KO;Pitx3GFP/GFP animals, compared to both the En1Het;Pitx3GFP/GFP and En1WT;Pitx3GFP/GFP animals (*** = P<0.01, n = 3/4).</p

    SHH expression does not overlap with the majority of the TH<sup>+</sup> area of the midbrain.

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    <p>(<b>A</b>) <i>In situ</i> hybridization of <i>Shh</i> in comparison to TH-DAB shows expression in the ventricular zone at E12.5. However, expression of <i>Shh</i> is stronger in the BP than in the FP of the embryonic midbrain. <i>Shh</i> expression seems to end at the border with the TH expressing area (<b>1</b>). (<b>B</b>) At E11.5 and E12.5 SHH expression does not overlap with most of the TH<sup>+</sup> cells in both rostral and caudal regions of the TH<sup>+</sup> area. However, at the most lateral parts of the TH expressing area some overlap seems to exist with SHH (<b>1–2</b>).</p

    Quantification of the number of GFP-positive neurons present in the midbrains of multiple <i>En1</i>-/<i>Pitx3</i>-mutants at E14.5.

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    <p>(A) Schematic representation of the isolation of midbrain and R1, and subsequent FAC-sorting setup at E14.5 to be used for quantification of number of GFP-positive neurons. (B) At E14.5, ~15000 GFP-positive mdDA neurons were sorted from control, En1WT;Pitx3GFP/GFP and En1Het;Pitx3GFP/GFP animals. In contrast, only ~5000 GFP-positive mdDA neurons were present in the En1KO;Pitx3GFP/GFP midbrain/R1 (** = P<0.01, n = 3/4).</p

    Cells of the mdDA system seem to originate in the FP and at the FP-BP boundary and are positioned along-side and perpendicular to radial glia.

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    <p>Radial positioned neurons can be detected from E11.5 onwards. TH<sup>+</sup> neurons appear to originate in the FP and at the FP-BP boundary (white arrowheads in overview) and are positioned along-side radial glia (white arrowheads) (1–2). At E11.5 some radial positioned neurons can be detected rostrally, but most are present in the caudal part of the midbrain. When TH<sup>+</sup> neurons reach the ventral part of the midbrain, most neurons are positioned tangential (white arrows), suggesting migration to more lateral and rostral regions (3–4 and 3′, 3", and 4′). A: Anterior; P: Posterior.</p

    Schematic representation of roles of <i>En1</i> and <i>Pitx3</i> in the programming of the rostral-caudal identity of mdDA neurons.

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    <p>(A) In wild-type midbrain <i>Nurr1</i> initiates the development of mdDA differentiation, <i>Pitx3</i> promotes <i>Ahd2</i> expression and represses <i>En1</i> in rostral midbrain, whilst <i>En1</i> promotes <i>Pitx3</i> and <i>Cck</i> expression. The mdDA neuronal pool includes rostral-coded and caudal-coded neurons. (B) In <i>Pitx3</i>-ablated animals <i>Nurr1</i> initiates the development of mdDA differentiation, though <i>Ahd2</i> expression is lost, and the inhibition of <i>Pitx3</i> on <i>En1</i> is lifted, thus <i>En1</i> and subsequently <i>Cck</i> are up-regulated. The mdDA neuronal pool includes only caudal-coded neurons. (C) In <i>En1</i>-ablated animals <i>Nurr1</i> initiates the differentiation of mdDA progenitors, though <i>Cck</i> expression is lost, and <i>Pitx3</i> expression in the rostral midbrain is not initiated, thus <i>Ahd2</i> expression is lost as well. The mdDA neuronal pool includes only non-coded neurons. (D) In double En1KO;Pitx3GFP/GFP animals elevated levels <i>Nurr1</i> promotes the differentiation of mdDA progenitors, though <i>Cck</i> and <i>Ahd2</i> are lost.</p

    TH<sup>+</sup> cells in the midbrain co-localize at both E12.5 and E14.5 with β-galactosidase, in a BATGAL expressing transgenic mouse line.

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    <p>A) Model of the transgenic BATGAL mouse line. β-galactosidase is expressed by binding of stable β-catenin to the TCF/LEF binding sites in the promoter of the <i>LacZ</i> gene. When β-catenin is activated, the <i>LacZ</i> gene in-cooperated in the genome is transcribed and β-galactosidase expressed. (B) At E12.5 β-galactosidase expression overlaps with TH both lateral and medial in the mdDA system (1–4). At E14.5 no β-galactosidase staining is detected lateral. However, medial many mdDA neurons can be detected to express β-galactosidase (5–8). Indicating that canonical WNT-signaling is or has been present in mdDA neurons. A: Anterior; P: Posterior.</p
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