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

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，以及这些神经元是如何组装成功能性网络的，仍然是一个高度研究的问题，答案尚不清楚。深入了解这是如何实现的，有望使我们能够研究不同神经元类型的功能（例如，通过获得对它们的遗传访问）以及打开大门，将干细胞编程为特定的神经元类型，以研究和治疗神经系统疾病。过去几十年来，在模式生物（如果蝇和小鼠）中的工作已经开始揭示导致不同神经元类型特化的潜在分子机制。在这个分子层次的最顶端是空间和时间程序，允许干细胞及其后代知道它们在空间和时间中的位置和时间。例如，位于基底前脑（BF）和纹状体的成年胆碱能神经元都是从腹侧端脑中称为内侧神经节隆起（MGE）的特定胚胎区域出生的，该区域还产生其他神经元类型的前体，例如GABA能神经元。除了空间上的限制，胆碱能神经元类型出生在重叠的时间窗口从E10-E13。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特异性。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。由于在有丝分裂后的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），因此本提案的目标是确定这些特化程序。了解不同的胆碱能神经元类型是如何被指定的将使我们开始了解它们的功能。事实上，大脑中的胆碱能神经元通过调节不同的大脑回路来调节神经认知功能，如记忆、注意力和奖励。这些神经元的功能障碍与许多神经系统疾病有关，包括帕金森病和阿尔茨海默病。在目标1中，我将注释8个成人（P30）胆碱能神经元簇，我假设它们代表了位于纹状体不同部位的2种中间神经元类型和靶向不同脑区的投射神经元。在目标2中，我将通过收集和分析来自E10-E13的胆碱能前体来定义导致不同胆碱能类的发育程序，我将通过从我们的P30数据集回溯时间来注释。在目标3中，我计划使用现有的方法和开发新的策略来评估候选因子在指定胆碱能命运中的功能。这些操作的结果将通过表征簇特异性标志物的表达、投射模式和转录组谱的变化来确定。这项研究将为产生旨在靶向和操纵不同胆碱能神经元进行功能和行为研究的遗传策略奠定基础。

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.