Research On The Heterogeneity Of Zebrafish Liver Cells Based On Single Cell Sequencing

With the advantages of transparent embryo,in vitro fertilization,easy observation,rapid reproduction and small individual,zebrafish(Danio rerio)is widely used in the research fields of pharmacology,toxicology,developmental biology,immunology,molecular biology and other fields.Zebrafish liver is highly similar to human liver in cell phenotype,development process and functional mechanism.It can be used as a model organism for the study of liver diseases.So far,the studies of zebrafish liver were focused on the study of single gene level and transcriptome at the level of cell population.Studies based on cell population level often ignored the heterogeneity between cells and covered up the characteristics of specific cell types,which can be made up by single-cell RNA sequencing(sc RNA-seq).At present,single cell sequencing technology has been applied to the study of human and mouse liver,which provides valuable information for clarifying liver function and diseases.Analyzing the cell composition and gene expression characteristics of healthy adult zebrafish liver at the single-cell level is of great significance for the subsequent study of liver diseases in adult zebrafish models.In this study,we used high-throughput single-cell transcriptome sequencing technology to draw the single-cell transcriptome map of healthy adult zebrafish liver,which provides an important reference for the follow-up application of zebrafish liver model in liver growth,development,metabolism and disease.The main contents and results of our study are as follows:The zebrafish liver was sequenced by single-cell transcriptome,and the sequencing libraries were quality controlled,filtered and integrated.Finally,4,268cells were retained for subsequent analysis.After clustering and cell type annotation,a total of 10 cell types were obtained,including Hep 1,Hep 2,Hep 3,T cells,neutrophils/monocytes,non-inflammatory macrophages,inflammatory macrophages,eosinophils,B cells and erythrocytes.And by comparing the proportion of the same cell type in different samples,it is found that the relevant data are not affected by the batch effect.In addition,this study found that the cell types identified in zebrafish liver are very similar to mammals,which proves the conservation of vertebrate liver function.Three hepatocyte subclusters in zebrafish liver were re-clustered and annotated,and six hepatocyte subclusters were obtained finally.To further compare the heterogeneity between different hepatocyte subclusters,GO enrichment analysis was carried out for the differential genes of these six cell subclusters.And it was found that a hepatocyte subcluster(CL5)was highly enriched in alcohol metabolism pathway,PPAR signaling pathway,lipid metabolism pathway and detoxification pathway,indicating that CL5 plays an important role in lipid synthesis and metabolism,and has the potential to be used as a target cell for research on fatty liver diseases in zebrafish.At the same time,based on marker genes,two macrophage subclusters with different transcriptional characteristics,inflammatory macrophages and non-inflammatory macrophages,were also identified in zebrafish liver.And through the comprehensive comparative analysis of the gene expression pattern of inflammatory macrophages and non-inflammatory macrophages,it is found that the up-regulated pathways in inflammatory macrophages which are mainly related to inflammatory response,including IFN-γsignaling pathway and toll-like receptor signal pathway,while the highly enriched functional pathways in non-inflammatory macrophages are mainly related to immune tolerance,including complement activation pathway,oxidative phosphorylation pathway,electron transfer activity pathway,fatty acid binding pathway and intercellular recognition related pathway.Liver T cells play an important role in liver immune microenvironment.This study analyzed the T cell subclusters in zebrafish liver,mainly focusing on CD4~+T cells and CD8~+T cells.Based on different marker genes,six CD8~+T subclusters(CD8-rorc,CD8-ccl38.6,CD8-mcm4,CD8-mki67,CD8-gzmk and CD8-lta)were annotated.Through pseudotime analysis,it was found that these cell subsets followed a continuous development trajectory and gradually developed from functional cell type to exhausted cell type.While four CD4~+T cell subclusters(CD4-foxp3a,CD4-cd28,CD4-cebpb and CD4-mki67)were annotated.Pseudotime analysis showed that these subclusters also had a continuous development process.To analyze the interaction between fatty liver target cells and immune cells in zebrafish liver,this study used Cell Phone DB software to predict the potential ligand–receptor interactions between zebrafish hepatocyte subcluster CL5,non-inflammatory macrophages,inflammatory macrophages,CD4~+T cell subclusters and CD8~+T cell subclusters.The results showed that communications between hepatocytes subcluster CL5 and immune cells are bidirectional,And the relationship between macrophages and hepatocyte CL5 is the closest,indicating that macrophages may play an important role in immune regulation in the progression of fatty liver diseases.