Generation of interneurons derived from ES cells using inducible expression of tr

描述(申请人提供)：前脑GABA能中间神经元是大脑皮层抑制的主要来源，通过平衡皮质回路中的兴奋，在大脑功能的几乎各个方面发挥关键作用。中间神经元约占皮质神经元的20%，根据其神经化学标志物、连通性和生理特性被分为不同的亚型。然而，人们对产生不同亚型中间神经元的遗传和分子机制知之甚少。皮质中间神经元的异常发育和功能与几种主要的神经和精神疾病的病理生物学有关，包括精神分裂症、自闭症、癫痫和许多其他疾病。缺乏一种有效的机制来产生、收集和选择中间神经元，这极大地阻碍了我们研究中间神经元在疾病病因中的作用，以及分析它们作为基于细胞的治疗的潜力。目前从胚胎干细胞获得中间神经元的方法受到中间神经元比例较小以及生长抑素表达(SST+)中间神经元对其他亚群的偏向产生的阻碍。在这项提议中，我们希望增加ES来源的中间神经元的总体产量，并特别丰富小白蛋白表达(PV+)中间神经元。决定命运的关键转录因子的强制表达可以引导干细胞分化为特定的细胞类型。转录因子Nkx2.1由皮质间神经元祖细胞表达，是确定中间神经元亚型所必需的。我们正在寻求的一种策略是在ES细胞中诱导表达Nkx2.1，该细胞也包含一个关于神经元间命运的有丝分裂后报告(Lhx6：GFP)。另一种策略是利用Nkx2.1：mChery；Lhx6：GFP ES系，使我们能够专门分离Nkx2.1+祖细胞和新有丝分裂后Lhx6+细胞，这可能有助于丰富PV+中间神经元。我们将收集GFP+细胞并将其移植到新生小鼠的皮质中，以证实这些细胞在体内表达具有功能中间神经元特征的神经化学和电生理特性。在建立了产生高比例SST+和PV+中间神经元的不同方案后，我们将收集从这些不同条件下获得的GFP+细胞的RNA，并进行RNA-SEQ分析，以确定转录组的特征，并识别决定中间神经元命运的新的亚群特异性基因。总而言之，这项建议中概述的实验应该会增加我们产生ES细胞来源的中间神经元的能力，特别是产生特定亚型的中间神经元。这些结果将极大地帮助研究中间神经元的发育，并提高我们将中间神经元用作基于细胞的治疗疾病的能力。

DESCRIPTION (provided by applicant): Forebrain GABAergic interneurons are the primary source of inhibition in the cerebral cortex and play crucial roles in nearly every aspect of brain function by balancing excitation in cortical circuits. Interneurons comprise approximately 20% of cortical neurons and are divided into different subtypes based on their neurochemical markers, connectivity and physiological properties. However, very little is known about the genetic and molecular mechanisms that give rise to different subtypes of interneuron. The abnormal development and function of cortical interneurons has been implicated in the pathobiology of several major neurological and psychiatric disorders, including schizophrenia, autism, epilepsy, and numerous other diseases. The lack of an efficient mechanism for the production, collection and selection of interneurons has greatly hindered our ability to study both the role of interneurons in disease etiologies, as well as analyze their potential as cell based therapies. The current protocol to derive interneurons from embryonic stem (ES) cells is handicapped by the small percentage of interneurons produced and the biased production of somatostatin- expressing (Sst+) interneurons over other subgroups. In this proposal, we hope to enhance the overall production of ES-derived interneurons and specifically enrich for parvalbumin-expressing (PV+) interneurons. Forced expression of key fate-determining transcription factors can direct the differentiation of stem cells into specific cell types. The transcription factor Nkx2.1 is expressed by cortical interneuron progenitors and is required for the specification of interneuron subtypes. One strategy we are pursuing is to inducibly express Nkx2.1 in ES cells that also contain a post-mitotic reporter for interneuron fate (Lhx6:GFP). Another strategy is to utilize an Nkx2.1:mChery;Lhx6:GFP ES line that allows us to specifically isolate Nkx2.1+ progenitor cells and newly-postmitotic Lhx6+ cels, which could help enrich for PV+ interneurons. We will collect and transplant GFP+ cells into the cortex of neonatal mice to confirm that these cells express neurochemical and electrophysiological properties characteristic of functional interneurons in vivo. After establishing distinct protocols that produce a high percentage of Sst+ and PV+ interneurons, we will collect RNA from GFP+ cells obtained from these different conditions and perform RNA-seq analysis to characterize the transcriptome and identify novel subgroup-specific genes that determine interneuron fate. In sum, the experiments outlined in this proposal should increase our ability to produce ES cell-derived interneurons, and in particular, production of specific subtypes of interneurons. These results will significantly aid the study of interneuron development and advance our capabilities to use interneurons as cell-based therapies for the treatment of disease.