Abstract：
Single-cell transcriptomics is the current gold standard for global gene expression profiling, not only in mammals and model species, but also in non-model fish species. This is a rapidly expanding field, creating a deeper understanding of tissue heterogeneity and the distinct functions of individual cells, making it possible to explore the complexities of immunology and gene expression on a highly resolved level. In this study, we compared two single cell transcriptomic approaches to investigate cellular heterogeneity within the head kidney of healthy farmed Atlantic salmon (Salmo salar). We compared 14,149 cell transcriptomes assayed by single cell RNA-seq (scRNA-seq) with 18,067 nuclei transcriptomes captured by single nucleus RNA-Seq (snRNA-seq). Both approaches detected eight major cell populations in common: granulocytes, heamatopoietic stem cells, erythrocytes, mononuclear phagocytes, thrombocytes, B cells, NK-like cells, and T cells. Four additional cell types, endothelial, epithelial, interrenal, and mesenchymal cells, were detected in the snRNA-seq dataset, but appeared to be lost during preparation of the single cell suspension submitted for scRNA-seq library generation. We identified additional heterogeneity and subpopulations within the B cells, T cells, and endothelial cells, and revealed developmental trajectories of heamatopoietic stem cells into differentiated granulocyte and mononuclear phagocyte populations. Gene expression profiles of B cell subtypes revealed distinct IgM and IgT-skewed resting B cell lineages and provided insights into the regulation of B cell lymphopoiesis. The analysis revealed eleven T cell sub-populations, displaying a level of T cell heterogeneity in salmon head kidney comparable to that observed in mammals, including distinct subsets of cd4/cd8-negative T cells, such as tcrγ positive, progenitor-like, and cytotoxic cells. Although snRNA-seq and scRNA-seq were both useful to resolve cell type-specific expression in the Atlantic salmon head kidney, the snRNA-seq pipeline was overall more robust in identifying several cell types and subpopulations. While scRNA-seq displayed higher levels of ribosomal and mitochondrial genes, snRNA-seq captured more transcription factor genes. However, only scRNA-seq-generated data was useful for cell trajectory inference within the myeloid lineage. In conclusion, this study systematically outlines the relative merits of scRNA-seq and snRNA-seq in Atlantic salmon, enhances understanding of teleost immune cell lineages, and provides a comprehensive list of markers for identifying major cell populations in the head kidney with significant immune relevance.
摘要：
单细胞转录组学是目前全球基因表达谱分析的黄金标准。不仅在哺乳动物和模型物种中，而且在非模型鱼种中也是如此。这是一个迅速发展的领域，对组织异质性和单个细胞的独特功能有了更深入的了解，使得在高度分辨的水平上探索免疫学和基因表达的复杂性成为可能。在这项研究中,我们比较了两种单细胞转录组学方法，以研究健康养殖的大西洋鲑鱼（Salmosalar）头肾内的细胞异质性。我们比较了通过单细胞RNA-seq（scRNA-seq）测定的14,149个细胞转录组和通过单核RNA-Seq（snRNA-seq）捕获的18,067个核转录组。两种方法都检测到八种常见的主要细胞群：粒细胞，造血干细胞，红细胞,单核吞噬细胞,血小板,B细胞,NK样细胞,和T细胞。四种额外的细胞类型，内皮，上皮，肾间,和间充质细胞，在snRNA-seq数据集中检测到，但似乎在制备提交给scRNA-seq文库生成的单细胞悬浮液期间丢失。我们确定了B细胞内的其他异质性和亚群，T细胞,和内皮细胞，并揭示了造血干细胞分化为粒细胞和单核吞噬细胞群的发育轨迹。B细胞亚型的基因表达谱揭示了不同的IgM和IgT偏斜的静息B细胞谱系，并提供了对B细胞淋巴细胞生成调节的见解。分析揭示了11个T细胞亚群，鲑鱼头肾中的T细胞异质性水平与哺乳动物中观察到的水平相当，包括不同的CD4/CD8阴性T细胞亚群，如TCRγ阳性，祖先样，和细胞毒性细胞。尽管snRNA-seq和scRNA-seq均可用于解析大西洋鲑鱼头肾中的细胞类型特异性表达，snRNA-seq管道在识别几种细胞类型和亚群方面总体上更加稳健.虽然scRNA-seq显示出更高水平的核糖体和线粒体基因，snRNA-seq捕获了更多的转录因子基因。然而,只有scRNA-seq生成的数据对髓系内的细胞轨迹推断有用.总之,本研究系统地概述了scRNA-seq和snRNA-seq在大西洋鲑鱼中的相对优点，增强对硬骨鱼免疫细胞谱系的理解，并提供了用于识别具有显着免疫相关性的头肾中主要细胞群的标记物的全面列表。
