Isolation and interrogation of the transcriptional profile of pioneer neurons

先锋神经元转录谱的分离和询问

• 批准号: 9978397
• 负责人: Alex Nechiporuk
• 金额: $ 43.24万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978397
• 关键词: Actins; Address; Afferent Neurons; Axon; Behavior; Binding; Bioinformatics; Biological Models; Biology; CRISPR screen; Cell Line; Cells; Complex; Data; Development; Embryo; Environment; Exhibits; Filopodia; Fluorescent in Situ Hybridization; Future; Ganglia; Gene Expression Profiling; Genes; Genetic; Genetic Transcription; Growth; Growth Cones; Injury; Invertebrates; Knowledge; Label; Ligands; Molecular; Morphology; Natural regeneration; Nerve Growth Factor Receptors; Neurons; Pathway interactions; Peripheral; Peripheral Nervous System; Play; Population; Presynaptic Terminals; Proteomics; Protocols documentation; Receptor Activation; Receptor Protein-Tyrosine Kinases; Recovery; Role; Sensory; Sensory Ganglia; Signal Transduction; Specific qualifier value; System; Testing; Time; Transcript; Work; Zebrafish; axon growth; axon guidance; axonal pathfinding; base; differential expression; experimental study; genetic testing; glial cell-line derived neurotrophic factor; in vivo; innovation; interest; lateral line; mutant; nerve injury; nervous system development; neurodevelopment; neurotrophic factor; novel; programs; receptor; receptor internalization; response; retrograde transport; scaffold; single-cell RNA sequencing; therapeutic target; transcriptomics

Pioneer neurons are the first to extend axons to a particular region or target, acting as a guide and scaffold for “follower” axons. In most cases, pioneer neurons are essential in the developing central and peripheral nervous systems for the initial navigation to appropriate targets, proper follower axon pathfinding, and promoting follower axon outgrowth. Many studies have noted that pioneer neurons differ from followers in growth cone morphology and actin dynamics in multiple invertebrate and vertebrate model systems. These observations strongly argue that pioneer neurons possess a specific genetic program that controls distinct aspects of their growth cone morphology and behavior. Despite these critical roles and the unique axonal behavior of pioneer neurons, we still know little about 1) which genes are differentially expressed in pioneer versus follower neurons; and 2) how these transcriptional differences in turn promote specific pioneer neuron behaviors, such as enhanced axon outgrowth and protrusive activity in the growth cone. Our study will address this knowledge gap by identifying pioneer neuron-specific genes and testing their roles in axon growth. Our preliminary work has found that expression of a neurotrophin receptor Ret is highly elevated in a population of pioneering peripheral sensory neurons and is required for pioneer axon outgrowth. Furthermore, ret mutant pioneer axons display altered growth cone morphology, including reduced growth cone size and fewer filopodia. Thus, ret represents a unique marker of sensory pioneer axons. We will capitalize on this finding to isolate and interrogate a transcriptional profile of pioneer neurons. In Aim 1, we will use a single cell RNA-sequencing (scRNA-seq) to build a transcriptional profile of ret-positive pioneer neurons and identify genes that are enriched in the pioneer versus follower neuron subpopulations. In Aim 2, we will screen a list of genes from our scRNA-seq to identify candidates that play a role in growth cone dynamics and axon extension. In summary, our work will provide significant advancement by 1) generating a transcriptional profile of pioneer neurons during axon outgrowth to provide specific markers for studying pioneer neuron biology; and 2) testing the function of pioneer neuron-specific genes to identify new factors that promote pioneer axon growth in development.

先锋神经元是第一个将轴突延伸到特定区域或目标的神经元，充当着 “追随者”轴突。在大多数情况下，前驱神经元在发育中的中枢和外周是必不可少的。 最初导航到适当目标的神经系统，适当的跟随者轴突寻路，以及 促进跟随者轴突生长。许多研究指出，先锋神经元与跟随者在以下方面有所不同 多种无脊椎动物和脊椎动物模型系统中的生长锥形态和肌动蛋白动力学。这些 观察结果有力地证明，先锋神经元拥有一种特定的遗传程序，该程序控制着不同的 它们的生长锥形态和行为方面。尽管这些关键角色和独特的轴突 对于先锋神经元的行为，我们仍然知之甚少：1)哪些基因在先锋神经元中有差异表达 以及2)这些转录差异如何反过来促进特定的前驱神经元 表现为轴突突起增强和生长锥体内的突起活动。我们的研究将解决 通过识别先锋神经元特异性基因并测试它们在轴突生长中的作用，来解决这一知识差距。我们的 初步研究发现，神经营养素受体Ret的表达在一群 先锋外周感觉神经元，是先锋轴突生长所必需的。此外，ret突变体 先锋轴突显示生长锥体形态改变，包括生长锥体大小减小和数量减少 丝状伪足。因此，ret代表了感觉先锋轴突的独特标记物。我们将利用这一发现来 分离和询问先锋神经元的转录图谱。在目标1中，我们将使用单细胞rna测序(scrna-seq)来构建ret阳性前驱神经元的转录图谱，并识别 在先锋神经元亚群和跟随神经元亚群中含量丰富。在目标2中，我们将筛选来自 我们的scRNA-seq用于确定在生长锥动态和轴突延伸中发挥作用的候选基因。在……里面 总结，我们工作将通过1)生成先锋的转录简档来提供显著的进步 轴突生长过程中的神经元，为研究先锋神经元生物学提供特异性标记；以及2)测试 先锋神经元特异性基因在识别促进先锋轴突生长的新因素中的作用 发展。