Investigating V1 interneuron subtype diversity using embryonic stem cells

 描述(申请人提供)：成熟的神经系统包含数千个不同的神经元亚型，每个亚型都有专门的身份、连接和神经功能。 电路。人们对这种非凡的多样性是如何在发展过程中产生的知之甚少。了解神经元亚型多样化的过程将对神经回路如何组装以产生不同的行为提供重要的见解，并揭示为什么特定的神经元亚型在一些神经退行性疾病中选择性地脆弱。在这项提案中，我将重点介绍V1中间神经元(IN)的亚型多样化，IN是脊椎动物脊髓中的一类抑制神经元，对控制运动神经回路活动至关重要。在脊髓发育过程中，V1祖细胞结构域产生20多种不同的V1IN亚型，这提出了一个重要的问题，即同一祖细胞结构域如何衍生出不同的IN细胞类型。研究最多的V1IN亚型之一是Renshaw细胞(RC)，它提供运动神经元(MN)的经常性抑制。RCs是一种特别受关注的细胞类型，因为有证据表明在MN病肌萎缩性侧索硬化症(ALS)中RC复发抑制回路选择性受损。然而，控制V1祖细胞分化为RCS等特化亚型的分子机制目前尚不清楚。我们的实验室率先使用胚胎干细胞(ESC)来源的神经元来研究脊髓MN分化和亚型多样化的分子机制。在初步研究中，我发展和优化了胚胎干细胞向V1IN的分化，包括显示它们概括了正常的V1IN发育。重要的是，体外衍生的V1 INS表达V1亚型特异的分子标记，包括RC特异标记Calbindin(CB)。在本项目建议书中，我将利用体外分化系统来测试特定的 假设包括RCS在内的不同V1亚型的规范取决于神经发生的时间，其方式受Notch信号通路调节。我预计这些结果将(1)建立一个可通过实验获得的RCS体外模型，可用于研究它们在正常脊髓生理学和ALS等神经疾病中的作用；(2)对产生V1亚型多样性的分子机制提供前所未有的见解，这些知识可用于有效地将多能干细胞分化为与临床相关的细胞类型，用于疾病建模、药物发现和细胞替代治疗。

 DESCRIPTION (provided by applicant): The mature nervous system contains thousands of distinct neuronal subtypes, each with specialized identity, connectivity and function within neural circuits. How this remarkable diversity is generated during development is poorly understood. Understanding the process of neuronal subtype diversification will yield important insights into how neural circuits are assembled to produce distinct behaviors, as well as reveal why specific neuronal subtypes are selectively vulnerable in some neurodegenerative diseases. In this proposal, I will focus on the subtype diversification of V1 interneurons (IN), a class of inhibitor neurons in the vertebrate spinal cord that are essential for controlling motor circuit activity. During spinal cord development, the V1 progenitor domain produces more than two dozen distinct V1 IN subtypes, raising the important question of how diverse IN cell types are derived from the same progenitor domain. One of the best-studied V1 IN subtypes is the Renshaw cell (RC), which provides recurrent inhibition of motor neurons (MN). RCs are a cell type of special interest given evidence of selective impairment of the RC recurrent inhibitory circuit in the MN disease amyotrophic lateral sclerosis (ALS). However, molecular mechanisms controlling differentiation of V1 progenitors into specialized subtypes such as RCs are currently unknown. Our lab has pioneered the use of embryonic stem cell (ESC)-derived neurons for studying molecular mechanisms of spinal MN differentiation and subtype diversification. In preliminary studies, I developed and optimized differentiation of ESCs to V1 INs, including showing that they recapitulate normal V1 IN development. Importantly, in vitro-derived V1 INs express V1 subtype-specific molecular markers, including the RC-specific marker calbindin (Cb). In this project proposal, I will take advantage of the in vitro differentiation system to test the specific hypothesis that specification of different V1 subtypes, including RCs, is dependent on timing of neurogenesis in a manner regulated by the Notch signaling pathway. I anticipate that these results will (1) establish an experimentally accessible in vitro model of RCs that can be used to study their role in normal spinal physiology and in neurological diseases such as ALS; and (2) provide unprecedented insights into molecular mechanisms generating V1 subtype diversity, knowledge that can be used to efficiently differentiate pluripotent stem cells into clinically-relevant cell types for modeling disease, drug discovery, and cellular replacement therapy.