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（meso-diencephalic dopamine, mdDA）神经元的发育，依赖于孤核受体Nurr1与成对样同源盒转录因子Pitx3的协同调控作用。所有mdDA神经元均需Nurr1以维持酪氨酸羟化酶（Th, tyrosine hydroxylase）的表达及神经元存活，而对Pitx3的依赖仅见于将发育为黑质致密部（substantia nigra pars compacta, SNc）的mdDA神经元亚群。既往研究显示，Pitx3基因敲除（Pitx3-/-）胚胎无法表达视黄酸（retinoic acid, RA）合成酶Ahd2；正常生理状态下，Ahd2仅选择性表达于SNc中依赖Pitx3的多巴胺能神经元。在Pitx3-/-胚胎中恢复视黄酸信号通路后，研究发现SNc神经元分化为Th阳性神经元，以及胚胎发育全程的存活维持，均选择性依赖于视黄酸的存在。上述结果提示视黄酸在SNc神经元发育中发挥关键调控作用，但目前仍不明确其他受Nurr1与Pitx3调控的靶基因，是否也会以类似Th的方式依赖视黄酸信号通路。为筛选受Pitx3缺陷的mdDA神经元影响、且可通过胚胎时期视黄酸处理恢复表达的基因，本研究证实Delta样配体1（Delta-like 1）、多巴胺D2受体（D2R, Drd2）以及酪氨酸羟化酶（TH）受Pitx3与视黄酸信号共同调控，进而影响mdDA神经元的终末分化表型。此外，本研究发现，Ahd2介导的视黄酸信号调控仅为Pitx3下游级联反应的一个分支：囊泡单胺转运体2（Vmat2）、多巴胺转运体（Dat）、Ahd2（Aldh1a1）、同源盒基因En1、同源盒基因En2及胆囊收缩素（CCK, cholecystokinin）的表达不受视黄酸处理影响，而是由Pitx3进行亚群特异性的调控。综上，本研究揭示了Pitx3调控的基因级联反应中多个视黄酸依赖与非依赖的调控环节，表明Pitx3可在多个分子层面参与mdDA神经元的亚群特化过程。本研究从胚胎第14.5天（E14.5）的Pitx3-/-与Pitx3+/+小鼠胚胎的解剖腹侧中脑组织中提取总RNA。每组实验样本包含3个Pitx3-/-小鼠的解剖腹侧中脑组织，与源自10个Pitx3+/+小鼠的解剖腹侧中脑组织提取的参考RNA进行杂交实验。