Isolation and interrogation of the transcriptional profile of pioneer neurons

先锋神经元是第一个将轴突延伸到特定区域或靶点的神经元，充当引导和支架， “追随者”轴突。在大多数情况下，先驱神经元在发育中的中枢和外周中是必不可少的。 神经系统的初始导航到适当的目标，适当的追随者轴突寻路， 促进跟随轴突的生长。许多研究已经指出，先锋神经元不同于追随者， 生长锥形态学和肌动蛋白动力学在多个无脊椎动物和脊椎动物模型系统。这些 观察强烈认为，先驱神经元拥有一种特定的遗传程序， 它们的生长锥形态和行为方面。尽管有这些关键作用和独特的轴突 先锋神经元的行为，我们仍然知道很少1）哪些基因在先锋神经元中差异表达 与跟随神经元; 2）这些转录差异如何反过来促进特定的先锋神经元 行为，如增强的轴突生长和生长锥中的增殖活性。我们的研究将解决 通过识别先驱神经元特异性基因并测试它们在轴突生长中的作用来填补这一知识空白。我们 初步研究发现，神经营养因子受体Ret的表达在患有神经营养因子缺乏症的人群中高度升高。 是先驱外周感觉神经元，并且是先驱轴突生长所必需的。此外，ret突变体 先驱轴突显示改变的生长锥形态，包括生长锥尺寸减小和生长锥数目减少。 丝状伪足因此，ret代表了感觉先驱轴突的独特标记。我们将利用这一发现， 分离并询问先驱神经元转录谱。在目标1中，我们将使用单细胞RNA测序（scRNA-seq）来构建ret阳性先驱神经元的转录谱，并鉴定 在先锋和跟随神经元亚群中富集。在目标2中，我们将筛选一系列基因， 我们的scRNA-seq来鉴定在生长锥动力学和轴突延伸中发挥作用的候选物。在 总之，我们工作将通过以下方面提供重大进展：1）产生先锋的转录谱 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异性标记;以及2）测试 先锋神经元特异性基因的功能，以确定新的因素，促进先锋轴突生长， 发展

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.