Transcriptional profiling of cutaneous Mrgprd free nerve endings and C-LTMRs

Cutaneous C-unmyelinated free nerve endings and hair follicles-innervating C-LTMRs convey two opposite aspects of touch sensation: a sensation of pain and a sensation of pleasant touch. The molecular mechanisms underlying these diametrically opposite functions are unknown. Here we used a mouse model that genetically marks C-LTMRs and the free nerve endings MRGPRD+ neurons in combination with fluorescent cell surface labeling, flow cytometry and RNA deep-sequencing technology. Cluster analysis of the RNA-Seq profiles of the purified neuronal subsets revealed 156 and 184 genes differentially expressed in MRGPRD-expressing neurons and C-LTMRs, respectively. 48 MRGPD- and 67 C-LTMRs-enriched genes were validated using a triple staining experiment approach and the Cav3.3 channel, found to be exclusively expressed in C-LTMRs, was validated using electrophysiology. Furthermore, our study greatly expands the molecular characterization of C-LTMRs and suggests that this particular population of neurons shares common transcriptional signatures with A and A low threshold mechanoreceptors. RNA profiles of FACS-sorted subsets of sensory neurons, namely Mrgprd+ neurons (DP, double-positive=IB4+GINIP+), C-LTMRs (IB4-GINIP+) and remaining cells (DN, double-negative=IB4-GINIP-) were generated by deep sequencing in duplicate using Illumina HiSeq 2000.皮肤C类无髓鞘游离神经末梢与支配毛囊的C类低阈值机械感受器（C-Low Threshold Mechanoreceptors, C-LTMRs）可传递两种截然相反的触觉感知：痛觉与愉悦触觉。介导这两种完全对立功能的分子机制迄今尚未阐明。本研究采用可遗传标记C-LTMRs与游离神经末梢MRGPRD阳性神经元的小鼠模型，结合荧光细胞表面标记技术、流式细胞术与RNA深度测序技术。对纯化得到的神经元亚群的RNA测序转录组谱进行聚类分析后，结果显示在表达MRGPRD的神经元与C-LTMRs中分别存在156个和184个差异表达基因。通过三重染色实验验证了48个MRGPD富集基因与67个C-LTMRs富集基因，并利用电生理学实验验证了仅在C-LTMRs中特异性表达的Cav3.3通道。此外，本研究极大拓展了对C-LTMRs的分子特征解析，同时提示该特定神经元群体与A类及A类低阈值机械感受器共享共同的转录特征。本研究通过Illumina HiSeq 2000平台进行双重复深度测序，获取了经荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）得到的感觉神经元亚群的转录组数据，所分选的亚群包括：Mrgprd阳性神经元（DP，双阳性=IB4+GINIP+）、C-LTMRs（IB4-GINIP+）以及剩余细胞（DN，双阴性=IB4-GINIP-）。