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扩展到特定区域或靶标的，充当指导和脚手架 “追随者”轴突。在大多数情况下，先锋神经元在发展中心和周边是必不可少的最初导航到适当目标的神经系统，适当的追随者轴突路径和促进追随者轴突的生长。许多研究指出，先锋神经元与关注者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。这些观察结果强烈认为，先锋神经元具有控制不同的遗传程序其生长锥形态和行为的各个方面。尽管这些关键角色和独特的轴突先驱神经元的行为，我们仍然对1）在先驱中表达哪些基因与追随者神经元相比； 2）这些转录差异又如何促进特定的先锋神经元行为，例如增强的轴突生长和生长锥中的突出活性。我们的研究将解决通过鉴定先锋神经特异性基因并测试其在轴突生长中的作用，这种知识差距。我们的初步工作发现，神经营养蛋白受体RET的表达高度升高开创性的外围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出改变的生长锥形态，包括降低生长锥大小和更少的丝状。这是RET代表感觉先锋轴突的独特标记。我们将利用这一发现隔离和询问先锋神经元的转录特征。在AIM 1中，我们将使用单个细胞RNA测序（SCRNA-SEQ）来构建RET阳性先锋神经元的转录曲线，并确定基因在先锋与追随者神经元亚群中富集。在AIM 2中，我们将筛选一个基因列表我们的SCRNA-SEQ确定在生长锥动力学和轴突扩展中发挥作用的候选者。总结，我们的工作将通过1）产生先锋的转录概况提供重大进步轴突生长过程中的神经元为研究先驱神经元生物学提供特定标记； 2）测试先锋神经特异性基因确定促进先锋轴突生长的新因素的功能发展。