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

人类的大脑由数十亿种不同类型的神经元组成。这种多样性是如何产生的，这些神经元是如何组装成功能网络的，这些问题仍然是高度研究的问题，答案尚不清楚。深入了解这是如何实现的，将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），并打开将干细胞编程为特定神经元类型以研究和治疗神经系统疾病的大门。在过去的几十年里，对模式生物（如果蝇和老鼠）的研究已经开始揭示导致不同神经元类型形成的潜在分子机制。在这个分子层次的最顶端是空间和时间程序，它允许干细胞和它们的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的一个特定胚胎区域产生的，称为内侧神经节隆起（MGE），该区域也产生其他神经元类型的前体，如gaba能神经元。除了受到空间限制外，胆碱能神经元类型在E10-E13重叠的时间窗口中诞生。因此，空间（MGE限制）和时间（E10-E13限制）程序的结合有助于胆碱能规范。Fishell实验室的初步工作发现，至少有8种不同的胆碱能神经元类型位于BF和纹状体中，但这些亚型在发育过程中是如何被指定的尚不清楚。由于不同胆碱能神经元类型的规范可能发生在有丝分裂后的MGE中（gaba能神经元的情况也是如此），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Aim 1中，我将注释8个成人（P30）胆碱能神经元簇，我假设它们代表位于纹状体不同部位的2种中间神经元类型和针对不同大脑区域的投射神经元。在目标2中，我将通过收集和分析E10-E13的胆碱能前体来定义导致不同胆碱能类别的发育计划，我将通过从我们的P30数据集中及时向后工作来注释。在Aim 3中，我计划使用现有的方法并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描述簇特异性标记的表达、投射模式和转录组谱的变化来确定。本研究将为产生针对不同胆碱能神经元的功能和行为研究的靶向和操纵遗传策略奠定基础。

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.