| Apostichopus japonicus is one of Holothuroidea,Echinodermata,which has an extensive coelom filled with coelomic fluid and coelomocytes and the coelom is located between digestive tract and the body wall ranging from the lime ring to the cloacal aperture.Meanwhile,there is a complex water-vascular system in A japonicus,which is also filled with coelomic fluid and coelomocytes.Generally,the coelom and water-vascular system are considered as the main sites for storing coelomocytes.Previous studies have shown that it is significantly different in the activities of immune enzymes in coelomic fluid supernatant and the composition of coelomocytes types between the water-vascular system and coelom.In this study,to further explore the correlation between the water-vascular system and coelom,a variety of substances are injected into coelom.Thus,the distribution of injected substances are detected in the coelom and water-vascular system(represented by polian vesicle)respectively to analyze the exchange of these substances.Next,the coelomocytes from coelom and water-vascular system are collected to analyze their subpopulations and the functions of each subpopulation by single-cell RNA sequencing technique(sc RNA-seq)through the differentially expressed genes of every subpopulations combined with their GO and KEGG enrichment analysis.In addition,we also analyzed the differences between groups of coelomic cells in the coelomic and aqueduct systems to explore whether it is different in the composition proportion and their functions of each subgroup between the coelom and water-vascular systemin A.japonicus.In this study,the distribution of injected substances in the coelom and the water-vascular system are detected to analyze the exchange of a variety of substances which are injected into coelom.The results showed that the coelomic fluid from coelom and polian vesicle(hereinafter referred to as "vesicle fluid")were stained into red at 6 hours after injection by beet red dye.At 6h and 12 h after GST-tag protein injection,a large number of proteins could be detected in the coelomic fluid,and there were also some proteins in the vesicle fluid.At 6 h and 12 h after injection by fluorescent microspheres,many fluorescent microspheres were observed in the coelomic fluid,but not in the vesicle fluid.In addition,at 6,12,24 and 72 h after injection by Escherichia coli and Vibrio splendidus,a large number of both bacteria were found in the coelomic fluid,but no bacteria were isolated in the vesicle fluid.In conclusion,this study shows that the water-vascular system and the coelom are not infinitely unblocked in A.japonicus.Soluble substances such as small molecules and biological macromolecules can be exchanged between the coelom and the water-vascular system,but the exogenous particles and bacteria cannot enter the water-vascular system via the coelom.In this study,we isolated the coelomocytes in the water-vascular system and coelom in A.japonicus andthe sc RNA-seq are carried outto obtain the relevant cell maps based on gene expression data of each cell.The results showed that a total of 14359 coelomic cells were collected in the water-vascular system and coelom.The coelomocytes were basically the same in the composition of clusters,which were clustered to 19 cell subpopulations.Each subpopulations accounted for 36.03%(Cluster 0)、15.19%(Cluster 1)、11.89%(Cluster 2)、8.49%(Cluster 3)、4.23%(Cluster 4)、6.60%(Cluster 5)、4.19%(Cluster 6)、3.74%(Cluster 7)、3.70%(Cluster 8)、0.77%(Cluster 9)、0.27%(Cluster 10)、1.10%(Cluster 11)、1.05%(Cluster 12)、0.92%(Cluster 13)、0.73%(Cluster 14)、0.42%(Cluster 15)、0.39%(Cluster 16)、0.14%(Cluster 17)、0.15%(Cluster18).Functional analysis of 19 subpopulations showed that most of clusters did not focus on specific functions,such as Cluster 0,which is involved in anti-infection,cell migration and adhesion in the immune system.Cluster 1 is involved in cell component biogenesis and cytoskeleton remodeling.Cluster 5 regulates genetic material and cell proliferation.Cluster 10 were associated with germ cell production and cell migration.Cluster 15 were correlated with cell proliferation regulation and stress response,and Cluster 17 were related to immune response and cell metabolism,etc.In addition,further analysis found cluster 4,9 and 14 haverelatively concentrated functions.These three clusters were analyzed by KEGG and GO enrichment,indicating that these three clusters were focused to perform certain specific biological functions.Cluster 4 is involved in coelomic cell growth regulation and metabolism.Cluster 9 participates in the immune defense response under the stimulations by bacteria or oxidative stress.The main function of cluster 14 is cytoskeletal rearrangement and chemotactic migration.The differences of the coelomocytes between coelom and the water-vascular system from each subpopulation are analyzed based on the sc RNA-seq.The results showed that it is significantly different in the cell proportion of the same clusters between coelom and the water-vascular system.The proportion of cells in CC sample were higher than PC sample in these 9 cell subpopulations(cluster 1,2,7,8,10,13,14,15 and 16),the difference multiples are different,mainly from 1 to 8 times.The proportion of cells in PC sample were higher than CC sample in these 9 cell subpopulations(cluster 0,3,4,5,6,9,11,12 and 17),the difference multiples are different,mainly from 1 to 6 times.Based on differences in gene expression between subpopulations at different sites,The results showed that the differentially expressed genes in some subpopulations were 790(Cluster 0),734(Cluster 1),538(Cluster 2),734(Cluster 3),165(Cluster 4),203(Cluster 5)and 229(Cluster 6),190(Cluster 7),241(Cluster 8),66(Cluster 13),and there were no or only one differential gene in these 8 cell subpopulations(cluster 10,11,15,17,18,9 and 12).We screened out cell subpopulations Cluster 2 and 5 with large differences in cell proportion and a large number of differential genes in the coelom and water-vascular system,the differential genes C3,Klkb1 and Gene00085 in Cluster 2 were significantly up-regulated.The differential genes C3,Gene00085,ITGA8 and MMP18 in Cluster5 were significantly upregulated by the analysis with software in the the water-vascular system compared with the coelom.Through bioinformatics analysis of sc RNA-seq library from various perspectives,it was concluded that Cluster 2 and 5 subpopulation may be involved in the immune related process of A.japonicus.However,cluster 2 and 5 intends to exert the function of immune defense through the classical pathway of complement activation and bypass activation pathway,while Cluster 2and 5 relies more on cell growth and migration for wound healing.In addition,we found that Cluster 2 also has phagocytic functions.No one or several functions werefocused through comprehensive analysis of differentially expressed gene,GO and KEGG enrichment in those clusters with few differentially expressed genes or similar coelomocytes proportion between coelom and water-vascular system.The result can be explained that the different proportion of coelomocytes in coelom and water-vascular system leads to the differences of subpopulation functions.The results of this study provide a basis for further understanding the fine classification and functional interpretation of coelomocytes in sea cucumber,and propose an important reference for revealing the connectivity between coelom and water-vascular system and the similarities and differences of coelomocytes functions between two sites. |
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