| Erythrocytes are one of the most abundant cell types in vertebrates.They are widely distributed in organism and are mainly responsible for transporting oxygen and carbon dioxide in the blood circulation.More and more studies have shown that erythrocytes also displayed other functions such as immune features.The precise regulation of erythrocytes differentiation process is the foundation for their functions.The development of erythrocyte is conserved among vertebrates,so zebrafish is often used as a model for the study of erythrocyte function and differentiation.However,the classification and functional characteristics of erythrocyte subtypes and the molecular mechanisms of developmental processes in zebrafish are still unclear.Moreover,the study of zebrafish erythrocyte maturation can also provide new insights for the study of mammalian erythrocyte enucleation.In this study,we analysis the of erythrocytes from single-cell sequencing data of head kidney,liver and spleen of zebrafish.We also analyzed the gene expression pattern during differentiation and maturation.Compared analysis between erythrocytes from zebrafish and human offered new ideas about the mechanism of erythrocyte enucleation and erythrocytes evolution.The specific research results are as follows:1.Subtype identification analysis provide new classification and function of erythrocytes.We identified 4 cell subtypes at different developmental stages(including progenitor erythrocytes,proliferating erythrocytes,division erythrocytes and mature erythrocytes)and 3 immune-related erythrocyte subtypes(including T cell related erythrocytes,myeloid-related erythrocytes,and interferon-related erythrocytes)and 1energy-related erythrocytes.Through the pseudo-time analysis of erythrocyte subtypes in four different developmental stages,we re-constructed the developmental trajectory of erythrocytes and analyzed the gene expression dynamics in different stages.Our results also suggest a various tissue distribution of erythrocytes in different states.Through functional enrichment and GSEA analysis,we found that early erythrocytes were in a state of active differentiation,while mature erythrocytes showed strong immune properties.Toll like receptor9 signaling pathway,Negative regulation of NF-?B transcription factor activity were significantly enrichment in mature erythrocytes.In addition,we also identified myeloid-related erythrocytes(lyz,lect2 l and tmsb4x),interferon-related erythrocytes(ifi46,isg15,stat1b)and T cell related erythrocytes(traj35,traj39,CT867973.2).The transcription factor prediction analysis proved that immune associated TF,such as spi1 b,irf8 and stat1,were significantly expressed in erythrocytes.2.Next,we conducted compare analysis between the human and zebrafish erythrocytes.We found the conservation expression of cell cycle and DNA replication genes(such as mcm7,tfdp1,rpa1)and the transcription factors involved in early erythrocyte differentiation(such as tal1,sox6,junb).Interestingly,it was found that RHO GTPase Effectors were significantly enrichment in human erythrocytes.In view of the difference between lower vertebrates and mammalian erythrocytes is that enucleation occurs specifically in mammalian erythrocytes.We further analyzed the signaling pathways such as RHO GTPases activate PKNs,RHO GTPases activate,CIT and ER-Phagosome pathway that affect the enucleation process.The results showed that the signaling pathway genes of cytokinesis and vesicle trafficking were low or not expressed in zebrafish erythrocytes.Similarly,the genes and transcription factors of WDR26,XPO7,E2F4 and FOXO3,which involved in chromatin condensation and enucleation,were also low or not expressed in zebrafish erythrocytes.Furthermore,zebrafish as a model for the study of erythrocyte diseases,we also analyzed the expression characteristics of 22 erythrocyte disease-related genes in zebrafish and human erythrocytes,which can provide candidate gene resources for erythrocyte disease research. |
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