基于单细胞转录组数据解析大鼠背根神经节在出生后发育中非神经元细胞变化特征

Deciphering the dynamic characteristics of non-neuronal cells in dorsal root ganglion of rat at different developmental stage based on single cell transcriptome data

摘要:

背根神经节(dorsal root ganglia, DRG)是重要的外周神经系统组成部分，是外周感觉传入中枢的枢纽。背根神经节在发育过程中神经元细胞及其基因表达的动态变化已有研究进行过单细胞转录组的解析，而关于非神经元细胞的动态变化尚无系统研究。为了探究出生后不同发育时间点大鼠DRG内非神经元细胞的变化，本研究选取10只7日龄(7 day, 7D)大鼠的DRG和3只3月龄(3 month, 3M)大鼠的DRG，制备单细胞悬液，使用10×Genomics平台进行测序，从单细胞水平解析了出生后发育中DRG非神经元细胞的转录图谱。结果表明，7D和3M各类非神经元细胞在细胞数目的分布比例上存在显著差异性。对拥有4个亚型的施旺细胞整体进行拟时分析，Ⅱ型施旺细胞是最早出现的施旺细胞亚型，Ⅲ型和Ⅳ型施旺细胞出现较晚。进一步对2个不同发育时间点细胞占比数差异显著的Ⅳ型施旺细胞进行了基因本体(gene ontology, GO)和京都基因与基因组百科全书(Kyoto encylopaedia of genes and genomes, KEGG)通路富集分析。从7D到3M的差异基因的GO分析结果表明，Ⅳ型施旺细胞的状态逐渐趋于稳定。KEGG分析结果发现酪氨酸代谢通路的显著上调影响了细胞内的信号转导，进而影响了细胞稳态的维持。从7D到3M，基因Col3a1、Col4a1显著下调，且与细胞外基质的构建密切相关，这表明Ⅳ型施旺细胞的细胞基质环境随着发育过程趋于稳定。上述结果提示Ⅳ型施旺细胞是一类趋于成熟且维持施旺细胞稳态的细胞。本研究关于DRG在发育过程中细胞类型和基因表达差异的分析结果为躯体感觉在发育过程中成熟机制的研究提供了重要参考信息。

Abstract:

Dorsal root ganglia (DRG) is an essential part of the peripheral nervous system and the hub of the peripheral sensory afferent. The dynamic changes of neuronal cells and their gene expression during the development of dorsal root ganglion have been studied through single-cell RNAseq analysis, while the dynamic changes of non-neuronal cells have not been systematically studied. Using single cell RNA sequencing technology, we conducted a research on the non-neuronal cells in the dorsal root ganglia of rats at different developmental stage. In this study, primary cell suspension was obtained from using the dorsal root ganglions (DRGs, L4–L5) of ten 7-day-old rats and three 3-month-old rats. The 10×Genomics platform was used for single cell dissociation and RNA sequencing. Twenty cell subsets were acquired through cluster dimension reduction analysis, and the marker genes of different types of cells in DRG were identified according to previous researches about DRG single cell transcriptome sequencing. In order to find out the non-neuronal cell subsets with significant differences at different development stage, the cells were classified into different cell types according to markers collected from previous researches. We performed pseudotime analysis of 4 types Schwann cells. It was found that subtype Ⅱ Schwann cells emerged firstly, and then were subtype Ⅲ Schwann cells and subtype Ⅳ Schwann cells, while subtype I Schwann cells existed during the whole development procedure. Pseudotime analysis indicated the essential genes influencing cell fate of different subtypes of Schwann cell in DRG, such as Ntrk2 and Pmp2, which affected cell fate of Schwann cells during the development period. GO analysis of differential expressed genes showed that the up-regulated genes, such as Cst3 and Spp1, were closely related to biological process of tissue homeostasis and multi-multicellular organism process. The down regulated key genes, such as Col3a1 and Col4a1, had close relationship with the progress of extracellular structure organization and negative regulation of cell adhesion. This suggested that the expression of genes enhancing cell homestasis increased, while the expression of related genes regulating ECM-receptor interaction pathway decreased during the development. The discovery provided valuable information and brand-new perspectives for the study on the physical and developmental mechanism of Schwann cell as well as the non-neuronal cell changes in DRG at different developmental stage. The differential gene expression results provided crucial references for the mechanism of somatosensory maturation during development.