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，通过平衡皮层回路中的兴奋，在大脑功能的几乎每个方面都起着至关重要的作用。中间神经元约占皮层神经元的20%，并根据其神经化学标记、连通性和生理特性分为不同的亚型。然而，关于产生中间神经元不同亚型的遗传和分子机制知之甚少。皮层中间神经元的异常发育和功能与几种主要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。缺乏有效的中间神经元的产生、收集和选择机制，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方法的潜力。目前从胚胎干（ES）细胞中获得中间神经元的方案受到产生的中间神经元百分比小和表达生长抑素（Sst+）的中间神经元比其他亚群偏倚的限制。在本研究中，我们希望提高es来源的中间神经元的整体产量，并特异性地富集表达parvalbumin （PV+）的中间神经元。关键的决定命运的转录因子的强制表达可以指导干细胞分化为特定的细胞类型。转录因子Nkx2.1由皮质中间神经元祖细胞表达，是中间神经元亚型分化所必需的。我们正在追求的一种策略是在胚胎干细胞中诱导表达Nkx2.1，这些胚胎干细胞也包含有丝分裂后神经元间命运报告基因（Lhx6:GFP）。另一个策略是利用Nkx2.1:mChery；Lhx6:GFP ES系，使我们能够特异性分离Nkx2.1+祖细胞和新的有丝分裂后Lhx6+细胞，有助于PV+中间神经元的富集。我们将收集GFP+细胞并将其移植到新生小鼠的皮层，以证实这些细胞在体内表达功能性中间神经元的神经化学和电生理特性。在建立了产生高百分比的Sst+和PV+中间神经元的不同方案之后，我们将从这些不同条件下获得的GFP+细胞中收集RNA，并进行RNA-seq分析，以表征转录组并鉴定决定中间神经元命运的新的亚群特异性基因。总之，本提案中概述的实验应该提高我们产生胚胎干细胞衍生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，并提高我们使用中间神经元作为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.