Coordination of olfactory receptor choice with guidance receptor expression and function in olfactory sensory neurons.
Coordination of olfactory receptor choice with guidance receptor expression and function in olfactory sensory neurons.
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DOI：
10.1371/journal.pgen.1007164
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发表时间：
2018-01
期刊：
影响因子：
4.5
通讯作者：
Raper JA
中科院分区：
文献类型：
--
作者：
Dang P;Fisher SA;Stefanik DJ;Kim J;Raper JA
嗅觉感觉神经元选择从大型基因库中表达单个气味受体 (OR)，并将轴突延伸到嗅球内可重复的、OR 特异性的位置。这个发育过程产生了大脑中气味体验的地形组织图。产生这种连接模式的轴突引导机制，以及协调 OR 选择和轴突引导受体表达的机制，尚不完全清楚。我们对斑马鱼幼虫新生的嗅觉感觉神经元 (OSN) 应用了单细胞 RNA 测序的强大方法来解决这些问题。通过单细胞 (SC) RNA-seq 生成 56 个单独的嗅觉标记蛋白 (OMP) 阳性感觉神经元的表达谱。我们发现，就像小鼠 OSN 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 靶向与感觉神经元选择表达其气味受体的 OR 进化枝有关。我们根据主要表达的 OR 的进化枝对每个成熟细胞进行分类。使用惩罚线性判别分析（PLDA）鉴定了三个进化枝相关类别中每一个类别中以较高水平表达的转录本。使用全基因组方法来识别最有可能具有引导相关活性的膜相关蛋白。我们发现选择表达来自特定进化枝的 OR 的 OSN 也表达潜在轴突引导基因和转录因子的特定子集。我们使用来自投射到单个原肾小球的转基因标记神经元子集的批量 RNA-seq 验证了对 OSN 一个分支的候选轴突引导基因的识别。使用荧光原位杂交证实了所选候选指导基因的差异表达模式。最重要的是，我们在本分析中确定的三个候选轴突引导基因（nrp1a、nrp1b 和 robo2）的敲除中观察到轴突定位错误。在每种情况下，在通常以高水平表达这些转录物的轴突子集中检测到靶向错误，而不是在低水平表达这些转录物的轴突中检测到靶向错误。我们的研究结果表明，特定的、功能性的、轴突引导相关的基因在 OSN 子集中表达，这些子集可以根据其 OR 表达模式进行分类。组装负责嗅觉的大脑回路是一项艰巨的任务。检测鼻上皮中气味的感觉神经元，即嗅觉感觉神经元，选择表达来自大型基因库的单一气味受体。选择特定气味受体的感觉神经元与选择不同受体的其他神经元混合。值得注意的是，所有选择特定气味受体的感觉神经元都将轴突投射到大脑中的同一目标区域，而那些选择表达其他气味受体的感觉神经元则将轴突投射到不同的目标区域。据推测，轴突靶向的这种特异性与感觉神经元亚群中引导相关基因的差异表达有关。有没有系统的方法来识别它们？在这里，我们使用单细胞 RNAseq 来表征单个嗅觉感觉神经元的转录谱。我们确定了候选指导相关基因，其表达与特定气味受体的同时表达相关。我们在功能丧失研究中表明，其中一些与指导相关的基因是指导嗅觉感觉神经元的特定子集所必需的。这种通用方法可能为系统识别基因打开大门，这些基因在发育中的神经回路组装过程中引导轴突到达其特定目标。
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. Assembling the brain circuitry responsible for our sense of olfaction is a formidable task. The sensory neurons that detect odorants in the nasal epithelium, the olfactory sensory neurons, choose to express a single odorant receptor from a large gene repertoire. Sensory neurons that choose a particular odorant receptor are intermixed with other neurons that have chosen different receptors. Remarkably, all the sensory neurons that choose a particular odorant receptor project axons to the same target region in the brain, while those choosing to express other odorant receptors project axons to different target regions. Presumably, this specificity in axon targeting is related to the differential expression of guidance related genes in subsets of sensory neurons. Is there a systematic method for their identification? Here we use single-cell RNAseq to characterize the transcriptional profiles of individual olfactory sensory neurons. We identify candidate guidance related genes whose expression is correlated to the simultaneous expression of particular odorant receptors. We show in loss of function studies that some of these guidance related genes are required for the guidance of specific subsets of olfactory sensory neurons. This general approach may open the door to the systematic identification of genes that guide axons to their specific targets during the assembly of developing neural circuits.
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影响因子：
64.8
作者：
Hong, Weizhe;Mosca, Timothy J.;Luo, Liqun
通讯作者：
Luo, Liqun
影响因子：
5.3
作者：
Cho, Jin Hyung;Prince, Janet E. A.;Cloutier, Jean-Francois
通讯作者：
Cloutier, Jean-Francois
影响因子：
16.2
作者：
Barth, AL;Dugas, JC;Ngai, J
通讯作者：
Ngai, J
DOI：
10.3791/1229
发表时间：
2009-03-25
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Brend, Tim;Holley, Scott A
通讯作者：
Holley, Scott A
影响因子：
4.6
作者：
Jhaveri, D;Saharan, S;Rodrigues, V
通讯作者：
Rodrigues, V