Nkx2.1对5-HT3AR/Sox6双阳性中间神经元发生发育的调控作用和机制

The balance between excitation and inhabitation is crucial for brain function. 5-HT3AR positive interneurons play a key role in constructing the cerebral neural network and keeping the balance between excitation and inhibition. It is widely accepted that their developmental defects could cause a variety of neurological disorders. However, the development and molecular mechanisms of 5-HT3AR positive interneurons are not fully understood. Our preliminary data demonstrates that there is a class of 5-HT3AR/Sox6 double-positive interneurons in the adult mouse brain, while there is a class of 5-HT3AR/ Nkx2.1 double-positive precursor cells in mouse medial basal ganglionic eminence and the preoptic area at the stage of embryonic development. Literature shows that Nkx2.1 is the upstream gene of Sox6. On the basis of these findings, we propose the hypothesis that 5-HT3AR/Sox6 double-positive interneurons may be derived from the medial ganglionic eminences and/or the preoptic area and Nkx2.1 could play a important role in the development of the 5-HT3AR/Sox6 double-positive interneurons. To substantiate this hypothesis, the present project aims to elucidate the development of the 5-HT3AR/Sox6 double-positive interneurons and to investigate the regulation of Nkx2.1 in their development using transgenic and conditional knockout mice. This project will shed light on new targets for drug discovery.

兴奋性和抑制性的动态平衡对大脑发挥正常功能至关重要。5-HT3AR+中间神经元是构建大脑神经网络，维持该动态平衡的关键细胞，其发育缺陷可引发多种神经系统疾病，但是目前对于5-HT3AR+中间神经元发生发育和相关调控机制尚不明确。申请者预实验发现：成年小鼠脑内存在一类5-HT3AR/Sox6双阳性中间神经元，而在胚胎发育时期，小鼠内侧基底神经节隆起和视前区存在5-HT3AR/Nkx2.1双阳性的中间神经元前体细胞。文献显示：Nkx2.1是Sox6的上游基因。据此提出假设：5-HT3AR/Sox6双阳性中间神经元可能起源于内侧基底神经节隆起或视前区，其发生发育可能受Nkx2.1的调控。为证实这一假设，本项目将以转基因和条件性基因敲除小鼠为研究对象，研究5-HT3AR+/Sox6+中间神经元的发生发育，探索Nkx2.1对其发生发育的调控机制,为研发治疗相关疾病理想药物提供新靶点。

表达5-羟色胺3A受体的（5-hydroxytrytamine-3A-receptor, 5-HT3AR）中间神经元是构建大脑神经网络，维持脑内兴奋性和抑制性动态平衡的关键细胞。5-HT3AR是唯一一类在大脑中间神经元表达的5-羟色胺（5-hydroxytrytamine, 5-HT）离子通道型受体，参与调控神经元的迁移、皮质电路的构建和皮质-丘脑通路的构建等多种发育过程。5-HT3AR+中间神经元的发育异常严重影响大脑神经网络的构建和功能，进而引发多种神经系统疾病。以往认为5-HT3AR+中间神经元主要来源于尾侧神经节隆起（CGE），且CGE来源的5-HT3AR+中间神经元不表达Sox6；5-HT3AR+中间神经元数量占大脑中间神经元总数的三分之一。通过本项目的实施，我们发现：.一、成年5-HT3AR-GFP小鼠皮层，海马以及纹状体中有部分的5-HT3AR+中间神经元表达Sox6。这部分的5-HT3AR/Sox6+双阳性中间神经元起源于MGE/POA，而并非DGE。.二、通过5-HT3AR-cre；Nkx2.1f/f获得Nkx2.1条件性基因敲除小鼠，我们发现：（1）皮层、海马和纹状体中5-HT3AR+中间神经元数量显著减少；（2）皮层中Lhx6的显著下调，而纹状体中Lhx8显著下调。由此可见，Nkx2.1通过调控Lhx6或Lhx8从而影响皮层和纹状体5-HT3AR+/Sox6+双阳性中间神经元的发生发育。.三、大脑中间神经元的起源区域比较复杂，随着我们实验的开展深入，我们发现纹状体中有几乎全部的ChaT+ 和5-HT3AR+中间神经元全部表达转录因子COUPTF-II，因此通过Nkx2.1-cre；COUPTF-IIf/f获得COUPTF-II条件性基因敲除小鼠，我们发现：纹状体中几乎不存在ChaT+和5-HT3AR+中间神经元，因此我们认为Nkx2.1通过调控转录因子COUPTF-II进而调控Lhx8，从而调控纹状体中ChaT+ 和5-HT3AR+中间神经元的发生发育的。.通过本项目的资助，分别在Development、Frontiers in Genetics和Behav Brain Res杂志发表论文各一篇，还有一篇准备投稿。

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