Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania Philadelphia, PA, United States of America.
Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, United States of America.
PLoS Genet. 2018 Jan 31;14(1):e1007164. doi: 10.1371/journal.pgen.1007164. eCollection 2018 Jan.
Olfactory sensory neurons choose to express a single odorant receptor (OR) from a large gene repertoire and extend axons to reproducible, OR-specific locations within the olfactory bulb. This developmental process produces a topographically organized map of odorant experience in the brain. The axon guidance mechanisms that generate this pattern of connectivity, as well as those that coordinate OR choice and axonal guidance receptor expression, are incompletely understood. We applied the powerful approach of single-cell RNA-seq on newly born olfactory sensory neurons (OSNs) in young zebrafish larvae to address these issues. Expression profiles were generated for 56 individual Olfactory Marker Protein (OMP) positive sensory neurons by single-cell (SC) RNA-seq. We show that just as in mouse OSNs, mature zebrafish OSNs typically express a single predominant OR transcript. Our previous work suggests that OSN targeting is related to the OR clade from which a sensory neuron chooses to express its odorant receptor. We categorized each of the mature cells based on the clade of their predominantly expressed OR. Transcripts expressed at higher levels in each of three clade-related categories were identified using Penalized Linear Discriminant Analysis (PLDA). A genome-wide approach was used to identify membrane-associated proteins that are most likely to have guidance-related activity. We found that OSNs that choose to express an OR from a particular clade also express specific subsets of potential axon guidance genes and transcription factors. We validated our identification of candidate axon guidance genes for one clade of OSNs using bulk RNA-seq from a subset of transgene-labeled neurons that project to a single protoglomerulus. The differential expression patterns of selected candidate guidance genes were confirmed using fluorescent in situ hybridization. Most importantly, we observed axonal mistargeting in knockouts of three candidate axonal guidance genes identified in this analysis: nrp1a, nrp1b, and robo2. In each case, targeting errors were detected in the subset of axons that normally express these transcripts at high levels, and not in the axons that express them at low levels. Our findings demonstrate that specific, functional, axonal guidance related genes are expressed in subsets of OSNs that that can be categorized by their patterns of OR expression.
嗅觉感觉神经元从大量的基因库中选择表达单一的气味受体（OR），并将轴突延伸到嗅球内可重复的、OR 特异性的位置。这个发育过程在大脑中产生了气味体验的拓扑组织图。产生这种连接模式的轴突导向机制，以及协调 OR 选择和轴突导向受体表达的机制，还不完全清楚。我们应用单细胞 RNA-seq 这一强大的方法，对幼斑马鱼幼虫中新出生的嗅觉感觉神经元（OSN）进行了研究，以解决这些问题。通过单细胞（SC）RNA-seq 为 56 个单独的嗅觉标记蛋白（OMP）阳性感觉神经元生成了表达谱。我们表明，正如在小鼠 OSN 中一样，成熟的斑马鱼 OSN 通常表达单一的主要 OR 转录本。我们之前的工作表明，OSN 的靶向与感觉神经元选择表达其气味受体的 OR 分支有关。我们根据其主要表达的 OR 分支对每个成熟细胞进行分类。使用惩罚线性判别分析（PLDA）识别在三个分支相关类别中的每一个中表达水平更高的转录本。使用全基因组方法鉴定最有可能具有导向相关活性的膜相关蛋白。我们发现，选择表达特定分支的 OR 的 OSN 也表达特定的潜在轴突导向基因和转录因子子集。我们使用针对投射到单个原基的转基因标记神经元的亚群的批量 RNA-seq 验证了我们对一个 OSN 分支的候选轴突导向基因的鉴定。使用荧光原位杂交技术证实了所选候选导向基因的差异表达模式。最重要的是，我们在三个候选轴突导向基因的敲除中观察到了轴突靶向错误：nrp1a、nrp1b 和 robo2。在每种情况下，在正常情况下这些转录本高水平表达的轴突亚群中检测到靶向错误，而在低水平表达这些转录本的轴突中则没有检测到。我们的发现表明，特定的、功能的、与轴突导向相关的基因在可以根据其 OR 表达模式进行分类的 OSN 亚群中表达。
