FJ001: Retinoic acid-dependent and -independent gene-regulatory pathways of Pitx3 in meso-diencephalic dopaminergic neurons

Development of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is only displayed by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we demonstrated that Pitx3-/- embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA-signaling in Pitx3-/- embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and TH are regulated by Pitx3 and RA signaling, influencing the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA-signaling represents only one aspect of the Pitx3 downstream cascade, since Vmat2, Dat, Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons. RNA was isolated from dissected ventral midbrains of E14.5 Pitx3-/- and Pitx3+/+ mouse embryos. 3 Experimental samples each consisting of 3 Pitx3-/- ventral midbrains were hybridized to reference RNA derived from 10 Pitx3+/+ ventral midbrains中脑-间脑多巴胺能神经元（meso-diencephalic dopamine, mdDA）的发育依赖孤核受体（orphan nuclear receptor）Nurr1与成对样同源框转录因子（paired-like homeobox transcription factor）Pitx3的协同调控。所有mdDA能神经元均需Nurr1以表达酪氨酸羟化酶（Tyrosine hydroxylase, Th）并维持存活，但对Pitx3的依赖性仅见于将发育为黑质致密部（substantia nigra pars compacta, SNc）的mdDA能神经元亚群。 此前本课题组已证实，Pitx3基因敲除（Pitx3-/-）胚胎无法表达视黄酸（retinoic acid, RA）合成酶Ahd2，而该酶通常选择性表达于SNc中依赖Pitx3的多巴胺能神经元。在Pitx3-/-胚胎中恢复RA信号通路后，研究发现SNc神经元的分化为Th阳性神经元以及胚胎发育全程的存活，均选择性依赖RA的存在。 尽管上述数据提示RA在SNc神经元中发挥重要的发育调控作用，但目前仍不明确其他受Nurr1与Pitx3调控的靶基因，是否也以类似Th的方式依赖RA信号通路。本研究旨在筛选在Pitx3缺陷型mdDA能神经元中表达异常、且经胚胎RA处理后可恢复正常表达的基因，最终证实Delta-like 1、多巴胺D2受体（Dopamine D2 receptor, D2R, Drd2）以及TH均受Pitx3与RA信号通路调控，进而影响mdDA能神经元的终末分化表型。 此外，本研究还发现，调控Ahd2介导的RA信号通路仅为Pitx3下游级联反应的一个环节：囊泡单胺转运体2（Vesicular monoamine transporter 2, Vmat2）、多巴胺转运体（Dopamine transporter, Dat）、Ahd2（乙醛脱氢酶1家族成员A1, Aldh1a1）、engrailed同源框蛋白1（engrailed 1, En1）、engrailed同源框蛋白2（engrailed 2, En2）以及胆囊收缩素（Cholecystokinin, Cck）的表达均不受RA处理影响，而是由Pitx3特异性调控其亚群特异性表达。 综上，本研究结果揭示了Pitx3调控的基因级联反应中多个RA依赖与非依赖的调控层面，表明Pitx3可在多个分子层面参与mdDA能神经元的亚群特化。 本研究从胚胎发育第14.5天（embryonic day 14.5, E14.5）的Pitx3-/-与野生型（Pitx3+/+）小鼠胚胎中，解剖分离腹侧中脑组织并提取总RNA。本研究设置3份实验样本，每份样本由3只Pitx3-/-小鼠的腹侧中脑组织组成，所有实验样本均与取自10只Pitx3+/+小鼠腹侧中脑组织的参照RNA进行杂交检测。