Research On The Regulatory Mechanism Of Cell Fate Decisions In The Macaque Cell Transcriptomic Atlas

Research background and purposeNon-human primates are the closest model organisms to humans in evolution,physiology,biochemistry,and pathology.Using Non-human primates as subjects provides high application value for the discovery of biological phenomena and mechanisms.It also provides more effective value when studying human diseases and clinical treatments.Constructing a comprehensive transcriptomic atlas of Macaca fascicularis,including of the peripheral blood,kidney,uterus,stomach,bone marrow,and esophagus,would provide important references for studying the physiological processes and tissue development.Cell fate decision is an important research field in terms of developmental biology.Even in mature or developed tissues,there are a large number of stem cells.Revealing the cell fate determination mechanism can help us better understand the stem cell transformation in the development and differentiation,and mature cell reprogramming and dedifferentiation during regeneration.Finally,our goal is to explain how tissues are formed and maintain their homeostasis.In this project,we identified the stem cells in the hematopoietic system and esophagus and constructed their differentiation trajectories.We explored the key transcription factors(TFs)during the differentiation process and revealed the critical factors that regulate the cell fate determination.Methods(1)Construction of single-cell transcriptomic atlas of bone marrow,esophagus and other organsMacaca fascicularis cryopreserved tissue was used for single-cell/single-nucleus extraction,and sc RNA-seq library preparation were performed using droplet microfluidic technology(i Drop)and followed by sequencing.The sequencing data was subjected to quality control,filtering,genome sequence alignment,and annotation to obtain single-cell gene expression data,and then data dimensionality reduction,clustering,and cell type annotation were performed.Next,identify marker genes of each cell type in bone marrow,esophagus,and other organs.(2)Identifying key TFs and regulatory mechanisms of stem cell differentiationWe annotated and classified different stem cell types and cell states according to the reported stem cell marker genes,and calculated the differentially expressed genes(DEGs)of each cell type.DEGs were enriched by GO ontology to identify functional alteration.The TF regulatory network was constructed using SCENIC software.We calculated the expression relationships and regulatory modules to identify TF modules with high connectivity.To identify key TFs whose expression change coordinates with transcriptional rates during differentiation,stem cell differentiation trajectories were constructed by calculating the RNA velocity.Finally,GO ontology of the identified key TFs and their target genes was performed to illustrate their roles in the differentiation process.ResultsFirstly,we identified rare cell types in peripheral blood,kidney,uterus,stomach,bone marrow and esophagus of cynomolgus monkeys,including CD34+hematopoietic stem cells in the bone marrow,the basal cells and the suprabasal cells in the esophagus,and three cell states in the basal cells,and four cell states in the suprabasal cells.In the research on the differentiation of pluripotent hematopoietic stem cells(HSCs),we identified the transcriptional changes during differentiation,constructed TF regulatory networks,and found the regulatory relationship between GATA1 and EZH2 in the differentiation from pluripotent HSCs to erythroid cells.Both GATA1 and EZH2 were jointly regulated by YBX1.Levels of YBX1 expression accompanied with transcriptional rate in erythroid cell proliferation and state transition.With the decrease of YBX1 expression,large number of cells entered telophase.In the research on the state transition of esophageal basal stem cells,the expression of a key TF SOX6 was identified.The SOX6 functions in the maintenance of basal stem cells.Meanwhile,the expression of the TF TP63,which also plays a regulatory role in stem cells,was independently identified.There is a regulatory relationship between TP63 and SOX6,and it may promote the activation and differentiation of esophageal basal stem cells by regulating cell cycle progression.Conclusion1.The differentiation of HSCs into ERPs in the hematopoietic cells is regulated by the expression of YBX1.YBX1 interacts and regulates with GATA1 and EZH2.2.The expression of TP63 induces basal stem cells to transit from a quiescent state to an active proliferative state,and promotes differentiation by interacting with SOX6.3.Two key genes,YBX1 and TP63,regulated the rate of differentiation by regulating the timespan of cell cycle.