Mechanisms Regulating the Specification and Differentiation of Unique Types of Cholinergic Neurons During Development

项目摘要 人类大脑由数十亿种不同的神经元组成。这种多样性是如何产生的， 这些神经元组装成功能网络仍然是高度研究的问题，但答案不清楚。 深入了解这一点是如何实现的，这将使我们能够研究不同组织的功能。 神经元类型（例如，通过获得对它们的遗传访问）以及打开门编程干细胞进入 研究和治疗神经系统疾病。在过去几十年里， 生物，如果蝇和小鼠，已经开始解开潜在的分子机制， 导致不同神经元类型的特化。在这个分子层次的最顶端是空间的， 让干细胞及其后代知道它们在空间和时间中的位置和时间的时间程序。 例如，位于基底前脑（BF）和纹状体中的成年胆碱能神经元都是从一个神经元中产生的。 腹侧端脑中的特定胚胎区域称为内侧神经节隆起（MGE）， 还产生其他神经元类型的前体，例如GABA能神经元。除了在空间上 在E10-E13的重叠时间窗中产生受限的胆碱能神经元类型。因此 空间（MGE限制）和时间（E10-E13限制）程序的组合有助于胆碱能 规范. Fishell实验室的初步工作已经发现了至少8种不同的胆碱能神经元类型 位于BF和纹状体，但这些亚型在发育过程中如何被指定尚不清楚。为 在有丝分裂后，MGE中可能发生不同胆碱能神经元类型的特化（如 GABA能神经元的情况），本提案的目标是确定这些规范程序。 了解不同的胆碱能神经元类型是如何指定的，将使我们开始了解它们的神经元类型。 功能协调发展的事实上，大脑中的胆碱能神经元调节神经认知功能，如记忆，注意力， 并通过调节不同的大脑回路来获得奖励。这些神经元的功能障碍与许多神经系统疾病有关。 包括帕金森氏症和阿尔茨海默氏症在内的疾病。在目标1中，我将注释8个成人（P30）胆碱能神经元， 神经元簇，我假设这代表了两种位于纹状体不同部位的中间神经元类型 和投射神经元针对不同的大脑区域。在目标2中，我将定义领先的发展计划 通过收集和分析来自E10-E13的胆碱能前体， 通过从我们的P30数据集向后工作来注释。在目标3中，我计划使用现有的方法， 开发新的策略，以评估指定胆碱能命运的候选因子的功能。成果 将通过表征簇特异性标记物的表达、投影和标记物的表达来确定这些操作的有效性。 模式和转录组谱的变化。本研究将为产生遗传 策略，旨在针对和操纵不同的胆碱能神经元的功能和行为研究。

PROJECT SUMMARY The human brain is comprised of billions of diverse neuronal types. How this diversity is generated and how these neurons are assembled into functional networks remain highly researched questions with unclear answers. Obtaining a deep understanding of how this is achieved promises to allow us to study the functions of different neuronal types (e.g., by gaining genetic access to them) as well as open the door to program stem cells into specific neuronal types to study and treat neurological diseases. Work spanning the last few decades in model organisms, such as Drosophila and mouse, has begun to unravel the underlying molecular mechanisms that lead to the specification of different neuronal types. At the very top of this molecular hierarchy are spatial and temporal programs that allow stem cells and their progeny to know where and when they are in space and time. For example, adult cholinergic neurons located in the basal forebrain (BF) and striatum are all born from a specific embryonic domain in the ventral telencephalon called the medial ganglionic eminence (MGE), which also produces precursors for other neuronal types such as GABAergic neurons. In addition to being spatially restricted, cholinergic neuronal types are born in overlapping temporal windows from E10-E13. Thus, the combination of spatial (MGE restricted) and temporal (E10-E13 restricted) programs contribute to cholinergic specification. Preliminary work in the Fishell lab has uncovered at least 8 distinct cholinergic neuronal types located in the BF and striatum but how these subtypes are specified during development is not known. As the specification into different cholinergic neuronal types likely occurs in the MGE upon becoming postmitotic (as is the case for GABAergic neurons), the goal of this proposal is to determine these specification programs. Understanding how different cholinergic neuronal types are specified will allow us to begin to understand their functions. Indeed, cholinergic neurons in the brain modulate neurocognitive functions such as memory, attention, and reward by regulating diverse brain circuits. Dysfunction of these neurons is linked to many neurological disorders, including Parkinson's and Alzheimer's diseases. In Aim 1, I will annotate the 8 adult (P30) cholinergic neuronal clusters, which I hypothesize represent the 2 interneuron types residing in different parts of the striatum and projection neurons targeting distinct brain areas. In Aim 2, I will define the developmental programs leading to different cholinergic classes by collecting and analyzing cholinergic precursors from E10-E13, which I will annotate by working backwards in time from our P30 dataset. In Aim 3, I plan to use existing methods and develop new strategies to assess the function of candidate factors in specifying cholinergic fates. The outcomes of these manipulations will be determined by charactering the expression of cluster specific markers, projection patterns, and changes in transcriptome profiles. This study will build the foundation for generating genetic strategies aimed at targeting and manipulating different cholinergic neurons for functional and behavioral studies.