Single-cell Next Generation Sequencing (scNGS) facilitates analysis of gonadal single cells transcriptomes and revealed germ cell heterogeneity

The unraveling of heterogenous cell populations, — previously masked in bulk transcriptome measurements — are made possible through analysis of single-cell transcriptomics. The goals of this study are to examine single-cell NGS-derived gonadal cell transcriptome profiling and to evaluate heterogeneity of gonadal germ cells in avian species. We performed single-cell RNA sequencing using embryonic gonadal cells of HH stage 28 from male and female zebra finches. We identified 12,489 male transcripts and 10,046 female transcripts. We annotated 9 different cell types from a total of 20 clustered single cells and identified that primordial germ cells, which have been considered as single population, can be distinguishable into three subtypes. The three PGC subtypes were specifically enriched for genes with expression patterns related to germness or pluripotency, suggesting functional differences in PGCs according to the three subtypes. Our study represents the first single-cell RNA seq analysis of gonadal transcriptomes in zebra finch. Our results revealed the first heterogeneity and intrinsic changes of gonadal primordial germ cells, which have hitherto been considered as a single population in avian species. We conclude that single-cell RNA seq based transcriptome analysis would offers new perspectives on avian primordial germ cells. Overall design: Gonadal cells profiling of male and female zebra finches at single cell level

此前在批量转录组（bulk transcriptome）检测中被掩盖的异质性细胞群体解析，可通过单细胞转录组学（single-cell transcriptomics）分析得以实现。本研究旨在分析基于下一代测序（Next Generation Sequencing，NGS）的禽类性腺细胞转录组图谱，并解析禽类性腺生殖细胞的异质性。本研究以雌雄斑胸草雀HH stage 28期的胚胎性腺细胞为材料，开展单细胞RNA测序（single-cell RNA sequencing，scRNA-seq），共鉴定得到12489个雄性转录本与10046个雌性转录本。本研究从20个聚类单细胞中注释得到9种不同细胞类型，并发现此前被认为是单一群体的原始生殖细胞（primordial germ cells，PGCs）可被划分为3个亚型。这3个PGC亚型中，表达模式与生殖特性或多能性相关的基因显著富集，提示不同亚型的PGCs存在功能差异。本研究为首个针对斑胸草雀性腺转录组的单细胞RNA测序分析，其结果首次揭示了此前在禽类中被视为单一群体的性腺原始生殖细胞的异质性与内在特性变化。本研究认为，基于单细胞RNA测序的转录组分析将为禽类原始生殖细胞研究提供全新视角。整体实验设计：在单细胞水平开展雌雄斑胸草雀的性腺细胞转录组图谱分析。