Investigating V1 interneuron subtype diversity using embryonic stem cells

 描述（由申请人提供）：成熟的神经系统包含数千种不同的神经元亚型，每种亚型在神经元内具有专门的身份、连接和功能。 电路.人们对这种显著的多样性是如何在发育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产生重要的见解，并揭示为什么特定的神经元亚型在某些神经退行性疾病中选择性脆弱。在这个提议中，我将专注于V1中间神经元（IN）的亚型多样化，这是脊椎动物脊髓中一类对控制运动回路活动至关重要的抑制神经元。在脊髓发育过程中，V1祖细胞结构域产生了二十多种不同的V1 IN亚型，这就提出了一个重要的问题，即不同的IN细胞类型是如何从相同的祖细胞结构域衍生出来的。研究得最好的V1 IN亚型之一是Renshaw细胞（RC），其提供运动神经元（MN）的复发性抑制。RC是一种特别感兴趣的细胞类型，给出了MN疾病肌萎缩性侧索硬化症（ALS）中RC复发抑制回路选择性受损的证据。然而，控制V1祖细胞分化为特定亚型（如RC）的分子机制目前尚不清楚。 我们实验室率先使用胚胎干细胞（ESC）衍生的神经元研究脊髓MN分化和亚型多样化的分子机制。在初步研究中，我开发并优化了ESC向V1 IN的分化，包括显示它们重现了正常的V1 IN发育。重要的是，体外衍生的V1 IN表达V1亚型特异性分子标记，包括RC特异性标记钙结合蛋白（Cb）。在这个项目建议书中，我将利用体外分化系统来测试特异性 不同V1亚型（包括RC）的特化取决于Notch信号通路调节的神经发生时间。我预期这些结果将（1）建立一个实验上可获得的RC体外模型，可用于研究其在正常脊髓生理学和神经系统疾病（如ALS）中的作用;和（2）提供了对产生V1亚型多样性的分子机制的前所未有的了解，可用于有效地将多能干细胞分化为临床相关的细胞类型以用于疾病建模的知识，药物发现和细胞替代疗法。

 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.