| Objective:Mammalian megakaryocytes primarily settle in the yolk sac and liver during embryonic life and in organs such as the bone marrow and spleen during adulthood.They perform various important functions such as platelet production,immune regulation,and microenvironmental support.Recent studies have revealed that megakaryocytes also reside in the lung,and their molecular characteristics differ significantly from those of fetal liver and bone marrow megakaryocytes.Lung megakaryocytes are enriched for a range of genetic sets for innate immune functions such as inflammatory response and immune response,suggesting that lung megakaryocytes may play an important role in immune regulation.However,current knowledge of lung megakaryocytes is limited to the traditional description of platelet production and molecular characteristics,leaving important questions unanswered about the hematopoietic origin,developmental patterns,heterogeneity,and platelet-producing regulatory mechanisms of lung megakaryocytes.In this research,a lineage-tracing mouse model was used to clarify the hematopoietic origin of lung megakaryocytes,and single-cell transcriptome sequencing was used to clarify their molecular characteristics and heterogeneity to offer new perspectives on lung megakaryocytes.Methods:(1)To investigate the hematopoietic origin of lung megakaryocytes during development using the Cdh5-CreER;Rosa26tdTomato lineage tracing mouse model,and also to resolve the developmental dynamics of lung megakaryocytes in mice by in situ immunofluorescence and flow cytometric analysis.(2)Transcriptome sequencing of lung megakaryocytes at eight time points from E11.5 to 8 weeks by STRT-seq single-cell transcriptome sequencing technology to map the transcriptome of lung megakaryocytes during development and to resolve the changes in their molecular characteristics.(3)To resolve the heterogeneity of lung megakaryocytes using the Seurat single cell transcriptome analysis process and cluster analysis,and to compare the differences in heterogeneity between them and liver megakaryocytes.(4)Characterization of megakaryocyte immune subpopulations by differential gene comparison and GO functional annotation,and identification of megakaryocyte immune subpopulations by in situ immunofluorescence and flow cytometric analysis.(5)Identifying the molecular characteristics of niche subpopulations by differential gene comparison and GO functional annotation,and mining their potential functions through intercellular interactions and recipient-ligand pair analysis.(6)To compare the differences in ploidy between lung megakaryocytes and liver megakaryocytes using ploidy detection assays,and to resolve the differences in platelet production pathways and platelet production signaling pathways in lung and liver megakaryocytes by pseudotime analysis.(7)To compare the differences in platelet-producing subpopulations of lung and liver megakaryocytes by bioinformatics analysis to reveal the differences in platelet-producing functions between lung and liver megakaryocytes and to investigate the platelet-producing capacity of lung megakaryocytes and the properties of the platelets produced using the megakaryocyte in vivo transplantation technique.Results:(1)Lung megakaryocytes are derived from dual hematopoietic origins and persist during development.The Cdh5-CreER;Rosa26tdTomato lineage tracing mouse model was used to label blood cells from yolk sac hematopoiesis and hematopoietic stem cell hematopoiesis and to detect megakaryocytes in the lungs.Flow cytometry and in situ immunofluorescence staining of mouse lungs at different developmental stages showed that lung megakaryocytes persist during development.The difference in platelet production capacity between embryonic and adult lung megakaryocytes was investigated by sorting out embryonic and adult lung megakaryocytes for in vivo transplantation,and the results showed that embryonic lung megakaryocytes had a higher platelet production capacity than adult lung megakaryocytes.(2)The transcriptome characteristics of lung megakaryocytes show a dynamic change with development.Single-cell transcriptome sequencing and bioinformatics analysis of mouse lung megakaryocytes at different time periods showed that the transcriptome characteristics of lung megakaryocytes showed a certain dynamic pattern with development.After birth,the platelet production capacity was significantly reduced,while the immunomodulatory characteristics were significantly enhanced.At the same time,embryonic lung megakaryocytes were shown to have organogenesis characteristics,suggesting that they may play an important function in lung development.(3)The heterogeneous characteristics of lung megakaryocytes differ significantly from those of liver megakaryocytes.Single-cell transcriptome sequencing data of lung and liver megakaryocytes at different developmental periods were integrated,and seven megakaryocyte subpopulations were identified based on the expression of differential genes.The composition of heterogeneous subpopulations of lung and liver megakaryocytes was significantly different,with lung megakaryocytes dominated by subpopulations with platelet-producing,nichesupporting,and immune characteristics.At the same time,the proportion of heterogeneous subpopulations of lung megakaryocytes varied dynamically with development,with platelet-producing and niche-supporting subpopulations predominantly distributed before birth and the immune subpopulation predominating after birth.(4)Two immune megakaryocyte subpopulations with antigen-presenting and neutrophilic properties are present in the lung.Two subsets of immune megakaryocytes with antigen-presenting and neutrophilic properties were identified by differential gene analysis,and CD206 and CD 177 were identified as surface marker molecules for the two immune subsets,respectively.(5)The lung megakaryocyte niche-supporting subpopulation has high expression of genes regulating lung development.Results of differential gene and gene set enrichment analyses suggest that the lung megakaryocyte niche-supporting subpopulation overexpresses a range of genes that regulate lung development and have the potential to support lung development.Intercellular interaction analysis suggested that the lung megakaryocyte niche-supporting subpopulation has potential interactions with a variety of lung stromal cells.(6)Lung megakaryocytes have a platelet production mode that is not dependent on polyploidization.By performing ploidy assays on megakaryocytes,we found that lung megakaryocytes showed low ploidy characteristics throughout development.By extracting cyc subpopulations,polyploidization subpopulations,and platelet-producing subpopulations of lung and liver megakaryocytes separately for the pseudotime analysis,we found differences in the signaling pathways of platelet-producing pathways between lung megakaryocytes and liver megakaryocytes,suggesting that lung megakaryocytes have a platelet production mode that is not dependent on polyploidization.(7)A comparative analysis of platelet production by lung and liver-derived megakaryocytes.A comparison of the platelet-producing subpopulations of lung and liver megakaryocytes suggested differences in the platelet-producing capacity and molecular characteristics of platelet production between the two.The results showed that although both lung and liver megakaryocytes could produce platelets,the number of platelets produced by lung megakaryocytes was less than that of liver megakaryocytes.We further examined the platelets after transplantation and found that the molecular characteristics of platelets produced by lung megakaryocytes differed from those of platelets produced by liver megakaryocytes.Conclusion:In this study,we investigated the hematopoietic origin of lung megakaryocytes,the dynamic changes in transcriptome characteristics,and cellular heterogeneity during development,and found that lung megakaryocytes are contributed by both yolk sac hematopoiesis and hematopoietic stem cell hematopoiesis and persist during development.Furthermore,lung megakaryocytes undergo significant changes in molecular characteristics and cellular heterogeneity during development,as evidenced by a significant decrease in platelet production capacity and a significant increase in immune features.Cellular heterogeneity analysis identified two subpopulations of immune megakaryocytes with antigenpresenting and neutrophilic characteristics,as well as a lung megakaryocyte nichesupporting subpopulation that expresses genes relevant to lung development.In addition,we investigated the platelet production pathway of lung megakaryocytes and the molecular characteristics of the platelets they produce and found that lung megakaryocytes have a unique platelet production mode that is not dependent on polyploidization and that the molecular characteristics of platelets produced by lung megakaryocytes differ from those produced by liver megakaryocytes.This study provides important data for the study of lung megakaryocytes under physiological and pathological conditions,and also has important insights into the study of"organ-specific platelets". |
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