Isolation and interrogation of the transcriptional profile of pioneer neurons

先锋神经元转录谱的分离和询问

基本信息

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
项目类别：
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

项目摘要

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，充当引导和支架在大多数情况下，先锋神经元在中枢发育和外周发育中至关重要。神经系统用于初始导航到适当的目标，适当的跟随轴突寻路，以及许多研究指出先锋神经元与追随者神经元的不同之处在于。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。观察结果强烈表明，先锋神经元拥有控制不同的特定遗传程序。尽管有这些关键作用和独特的轴突，但它们的生长锥形态和行为方面。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差异表达与追随神经元；2）这些转录差异如何反过来促进特定的先锋神经元我们的研究将解决轴突生长增强和生长锥突出活动等行为。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弥补知识差距。初步研究发现，神经营养素受体 Ret 的表达在人群中高度升高。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。先驱轴突显示生长锥形态，包括生长锥尺寸减小和更少因此，ret 代表了感觉先锋轴突的独特标记。分离并询问先锋神经元的转录谱在目标 1 中，我们将使用单细胞 RNA 测序 (scRNA-seq) 构建 ret 阳性先锋神经元的转录谱并识别基因在先锋神经元亚群和跟随者神经元亚群中富集。在目标 2 中，我们将从中筛选基因列表。我们的 scRNA-seq 来识别在生长锥动力学和轴突延伸中发挥作用的候选者。总之，我们的工作将通过以下方式提供重大进展：1）生成先锋的转录谱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定标记；2) 测试先锋神经元特异性基因的功能，以确定促进先锋轴突生长的新因素发展。