Single-cell RNA-sequencing Analysis Reveals Hematopoietic Pathways In The Zebrafish Head Kidney

Hematopoiesis is one of the basic life-sustaining functions of vertebrates,which occurs every moment in the organism,and the cells produced by the hematopoietic function also play a vital role in the daily activities of the organism.As the secrets of hematopoiesis are gradually unearthed,more and more researches focus on how to realize the whole process of hematopoiesis in vitro.This research is almost directly equivalent to whether blood cells can be mass-produced in vitro,and most of the problems of blood-related diseases will be solved because of the results of this research.Zebrafish is a very commonly used experimental fish,and as a model organism,research related to it covers various aspects.Prior to this,there have been many studies related to zebrafish hematopoietic function and hematopoietic system,but most of them revolve around During the formation of the hematopoietic system from the zebrafish embryo to juvenile,there are relatively few studies on the head kidney,the main hematopoietic organ in adult zebrafish,and there is no systematic and clear conclusion on the molecular mechanism of hematopoiesis.In the study of zebrafish hematopoiesis,the process of endothelial hematopoietic transformation is also very important,which reveals the origin of the hematopoietic system.With the emergence of new sequencing technology in recent years,the research on vertebrate hematopoiesis has been further recognized,such as single cell sequencing technology,which is a sequencing technology that can obtain the genome,transcriptome and epigenome information in each cell.By using single-cell sequencing technology,we can clearly understand the roles and functions played by each cell in an organism.This study takes the head kidney of adult zebrafish as the research object,uses single cell RNA sequencing technology to obtain the cell atlas of the head kidney of adult zebrafish,and analyzes the exercise process of hematopoietic function in the head kidney of adult zebrafish.The specific research results are as follows:1.After clustering and cell annotation of all cells captured within the adult zebrafish head kidney,13 cell types were identified.It includes hematopoietic stem cells,hematopoietic progenitor cells,a special lymphoid progenitor cell,red blood cells of three different stages,neutrophils of two different stages,eosinophils,macrophages differentiated from monocytes,B lymphocytes,T lymphocytes and endothelial cells.We found and identified various cell types in zebrafish through their marker genes,and found more evidence for their identity in their corresponding gene expression lists.Through functional enrichment of genes,we further identified these cell types and discovered the functions they each may still perform.2.In order to explore the differentiation pathway of hematopoietic cells in the adult zebrafish head kidney,we re-extracted the hematopoietic stem cells and hematopoietic progenitor cells annotated in the first step,and clustered them again.Obtained cell subpopulations with differentiation tendencies toward lymphoid,myeloid,and erythroid,and through the pseudochronological analysis of these cell subpopulations and their corresponding blood cell types,we found the respective differentiation trends in the three differentiation directions.The time relationship of the emergence of different kinds of cells,and the key transcription factors of each differentiation node in each direction were found through the gene expression in this process in the quasi-chronological analysis.Here,we focus on the analysis of the relationship between lymphoid-directed progenitors annotated in all cells and stem progenitors found in this subgroup.We found that in the quasi-chronological results,the former did not appear later than we had guessed,but this cell type appeared almost at the same time as hematopoietic stem cells.After sorting out the results of the pseudo-chronological analysis in the above three directions,we speculated and mapped the differentiation process from hematopoietic cells to mature blood cells in the head kidney of adult zebrafish based on these results,and marked important transcription factors in each differentiation node.3.We found that in the adult zebrafish head kidney,although the endothelial cells we found still have the potential to differentiate into hematopoietic stem cells,hematopoietic stem cells seem to retain some characteristics of endothelial cells,but the endothelial hematopoietic transformation seems to be affected by factors such as the NOTCH pathway inhibition.In summary,this study used single-cell RNA sequencing technology and bioinformatics analysis to draw a complete cell map of zebrafish head kidney,analyze its hematopoietic process,and preliminarily analyze the feasibility of hematopoietic endothelial transformation in adult zebrafish head kidney.