Olfactory Sensory Neuron Diversity Beyond OR Genes in mice [scRNAseq]

How functional cellular heterogeneities are regulated is fundamental for understanding the molecular basis of complex organs. Olfactory sensory neurons (OSNs) are an ideal model to investigate the regulation of cellular heterogeneity. The one-neuron-one-receptor organization and topographical mapping ensure the detection and precise translation of odor signals to the central neural system. Besides the diversity of OR genes and other molecular guiding axon sorting processes, single-cell transcriptome analysis revealed an OSN subpopulation, defined by Cd36, a lipid receptor gene. The function study exhibited lipid odor identification was impaired in Cd36-deficient mice. In this study, we systematically depicted the transcriptome diversity, spatial distribution, and specific functions of Cd36+ OSNs in the mouse olfactory epithelium. The specific molecular features of Cd36+ OSN we revealed implemented the programmed cellular diversity may be driven by their olfaction function. Furthermore, with the integrative analysis of single-cell transcriptome and epigenome profiles, we revealed the cis and trans regulatory signatures in Cd36+ OSN and identified Tshz1 and Mef2 as the key regulators that may directly regulate and promote the expression of Cd36 and drive the cellular diversity of OSNs. Especially, we demonstrated that Tshz1 is expressed coordinately with the choices of ORs, earlier than the expression of Cd36, which indicates it may act as a pioneer factor that instructs the lineage-specific expression of Cd36 and other genes, eventually leading to the cellular diversity of Cd36+ OSN. Our results provide novel knowledge on the regulation mechanism of cellular diversity of complex organs. Single cell RNA-seq of the whole olfactory epithelium and FACS-enriched mature OSNs from 19-day or 8-week-year-old mice using 10x Genomics Chromium Single Cell 3 Reagent Kit V3细胞功能异质性的调控规律，是解析复杂器官分子机制的核心基础。嗅觉感觉神经元（Olfactory Sensory Neurons, OSNs）是研究细胞异质性调控的理想模型。其“一个神经元-一个受体”的组织模式与拓扑投射图谱，确保了气味信号的精准检测并向中枢神经系统传递。除嗅觉受体（Odorant Receptor, OR）基因的多样性及指导轴突分选的各类分子过程外，单细胞转录组分析还揭示了一类以脂质受体基因Cd36为标记的OSN亚群。功能实验证实，Cd36敲除小鼠的脂类气味识别能力存在缺陷。本研究系统解析了小鼠嗅觉上皮中Cd36阳性OSNs的转录组多样性、空间分布特征与专属功能。本研究揭示的Cd36阳性OSNs特异性分子特征，提示其程序化的细胞异质性可能由其嗅觉功能所驱动。此外，通过整合单细胞转录组与表观基因组图谱分析，本研究解析了Cd36阳性OSNs的顺式与反式调控特征，并鉴定出Tshz1与Mef2作为关键调控因子，可直接调控并促进Cd36的表达，进而驱动OSNs的细胞异质性。尤为重要的是，本研究证实Tshz1的表达与嗅觉受体基因的选择协同发生，且早于Cd36的表达，提示其可作为先锋因子，指导Cd36及其他基因的谱系特异性表达，最终促成Cd36阳性OSNs的细胞异质性。本研究结果为解析复杂器官的细胞异质性调控机制提供了全新的认知。本研究采用10x Genomics Chromium 单细胞3'端测序试剂盒V3，对19日龄及8周龄小鼠的全嗅觉上皮组织与经荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）富集的成熟OSNs进行了单细胞RNA测序。