| Objectives:The classic function of megakaryocytes(MKs)is to generate platelets,and at the same time,they can perform multiple functions such as hematopoietic microenvironment support and immune regulation.Recent advances in single-cell RNA sequencing(scRNA-seq)technology have revolutionized our understanding of the molecular characteristics and heterogeneity of MKs.However,the previous studies mainly focused on limited organs at a specific developmental stage,rather than a transcriptomic atlas of MKs across different organs during ontogeny.Thus,it is still unknown how molecular characteristics and heterogeneity of MKs evolves during ontogenesis.This study plans to explore the temporal and spatial dynamics of the entire megakaryocytic system in mouse at the single-cell level.Methods:(1)Systematic screening of organ colonization of megakaryocytes during ontogeny in mice and humans by flow cytometry and immunofluorescence staining.(2)The temporal and spatial evolution of molecular characteristics and heterogeneity of mouse megakaryocytes was deciphered using single-cell transcriptome sequencing technology.(3)The evaluation and comparation of platelet production capacity of megakaryocytes during mouse ontogeny were conducted by using mitochondrial fluorescent tracing mouse and megakaryocytes transplantation models.Results:(1)Megakaryocytes persist in multiple organs and show different dynamic changes with development:Through systematic screening of multiple organs during ontogeny in mice and humans,we found that the head and peripheral blood are the main resident organs of megakaryocytes in addition to the yolk sac,fetal liver,lung,bone marrow,and spleen in early embryonic development.Furthermore,we found that megakaryocytes in seven major colonizing organs persisted at specific developmental stages and exhibited distinct dynamics with development as detected by flow cytometry and quantitative analysis.(2)Megakaryocytes from different organs distinctly differentiate from each other:We performed single-cell transcriptome sequencing on megakaryocytes from seven major resident organs at different developmental stages,and found that the transcriptome characteristics of megakaryocytes from different organs are quite different,indicating that the organ microenvironment might play important roles in the functional specification of megakaryocytes.(3)Organ sources and developmental stages lead to significant megakaryocytes heterogeneity:Through the integrated analysis of the heterogeneity of megakaryocytes derived from different developmental stages of seven organs,we found that megakaryocytes were divided into seven subpopulations with different characteristics.The distribution and composition of subpopulations in different organs are significantly different,and the seven subpopulations show unique dynamic changes with development,indicating that the heterogeneity of megakaryocytes is associated with their organ sources and developmental stages.(4)mt subpopulation of megakaryocytes represents a new "thrombopoiesis-biased"subpopulation:By integrating analysis of the heterogeneity of megakaryocytes at different developmental stages in seven organs,we identified a new mtRNA-enriched mt subpopulation group.The expression level of mtRNA in the mt subgroup is organ and developmental specific.It displays relatively low expression in various organs during the embryonic period,and begins to increase significantly in the liver during the fetal period,and mainly occurs after birth especially highly expressed in bone marrow and spleen,the main resident organs of megakaryocytes.To distinguish the mt subpopulation,we introduced Mito-Dendra2 mitochondrial fluorescent tracer reporter mice.We confirmed that the expression intensity of Mito-Dendra2 fluorescent protein in this tracer mice can reflect mtRNA expression levels,and identified the mitohigh(mt)subpopulation is present in the adult bone marrow.The mitohigh subgroup of megakaryocytes has higher cell size,more cell granularity and higher ploidy level,and we further proved that the mitohigh subgroup megakaryocytes has higher platelet-producing potential in vitro and in vivo.(5)The mt and plt subpopulations in megakaryocytes represent "thrombopoiesisbiased" subpopulations at different developmental stages:We identified two"thrombopoiesis-biased" megakaryocytes by heterogeneity analysis and in vivo plateletproducing assays.The plt "thrombopoiesis-biased" subpopulation mainly exists in the early embryonic development,while the mt "thrombopoiesis-biased" subpopulation increases significantly from the fetal period,and is the main platelet-producing subpopulation after birth in mice.At the same time,the mt subpopulation has significantly different molecular characteristics related to platelet production from the plt subpopulation,and we confirmed that the mt subpopulation might has higher platelet producing ability in in vivo transplantation experiments.We speculated that the evolution of the two platelet producing modes represented by the mt "thrombopoiesis-biased" subpopulation and the plt "thrombopoiesis-biased" subpopulation is crucial for meeting platelet requirements at different developmental stages.(6)Three "immune-regulatory" megakaryocyte subpopulations were enriched with distinct immune cell gene sets:We found that immune characteristic megakaryocytes are also heterogeneous and can be divided into three immune characteristic megakaryocyte subpopulations,which enrich B cell/antigen presentation,macrophage,and neutrophil associated gene expression.B cell/antigen presentation characteristic immune subpopulation is mainly originated from spleen and lung,macrophage characteristic immune subpopulation is originated from lung,while neutrophil-characteristic immune subset is mainly derived from bone marrow.The three "immune-regulatory"megakaryocytes are mainly distributed in postnatal and dynamically increase with development,suggesting that megakaryocytes might exert different types of immune regulation function in different postnatal organs.(7)Megakaryocytes from embryonic head may have potential supportive roles in organ development:Heterogeneity analysis revealed that microenvironmental-supporting characteristic megakaryocytes(niche subpopulation)were mainly contributed by embryonic head megakaryocytes,which highly expressed classical microenvironmentalsupporting characteristic genes and enriched extracellular matrix and neurodevelopmental biological processes.Interaction analysis of head megakaryocytes with other cell types of the head further suggested that microenvironment-supporting characteristic megakaryocytes might play important roles in head development.(8)Identification and preliminary application of common marker genes of megakaryocytes:The common high-expression genes of each organ derived megakaryocytes were screened according to the gene expression levels,and the highly expressed genes were distinguished from the common genes of various nonmcgakaryocytcs,and the genes highly expressed in megakaryocytes compared with other cell types were screened as mcgakaryocyte-specific common marker genes.We screened a total of 53 megakaryocyte-specific common marker genes.This set of common marker genes can accurately distinguish megakaryocytes from non-megakaryocytes,and is expected to be further applied to the identification and characterization of small groups of megakaryocytes in different organs.Conclusions:This study identified developmental changes in megakaryocyte numbers in multiple organs,including the embryonic head,and profiles the spatiotemporal molecular atlas of the developing mouse megakaryocyte system from seven main resident organs by scRNA-seq.This identified the organ-specific functional properties of megakaryocytes and revealed the evolution of the molecular characteristics and platelet production patterns of megakaryocytes during ontogeny.Furthermore,the organ-common marker genes of megakaryocytes were identified,which are expected to be further used as important tools for the extensive identification of megakaryocytes.This contributed to understand the functional diversity and plasticity of megakaryocytes,and provided a theoretical basis for future platelet regeneration and clinical applications. |
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