Expression Analysis And Functional Characterization Of EpoR In Hematopoietic System

Erythropoietin(EPO)and its receptor,erythropoietin receptor(EpoR)are essential for the erythropoiesis.They are indispensable for erythroid progenitors and early erythroid precursors on proliferation and survival.EpoR has been traditionally thought as an erythroid specific gene.Notably,accumulating evidence suggests that EpoR is expressed well beyond erythroid cells,including macrophages,hematopoietic stem cells,B cells,megakaryocytes,endothelial cells,nerve cells and so on.However,the current understanding of EpoR expression and function on non-erythroid cells is far from clear.To this end,we newly designed and constructed an EpoR-tdTomato-Cre mouse model.In EpoR-tdTomato-Cre mice,the coding sequences of fluorescent protein tdTomato and Cre recombinase were knocked in at the stop code of EpoR,and a P2A sequence which can separate the proteins before and after it in the process of translation was inserted between EpoR and tdTomato as well as between tdTomato and Cre to ensure their efficient expression and functional integrity.Compared with the EpoReGFPcre mouse model constructed in previous studies,the EpoR-tdTomato-Cre mouse model has many advantages:First,this mouse model uses the high brightness fluorescent protein tdTomato,which can efficiently identify cells with low expression of EpoR;second,this mouse model overcomes the problem of haploinsufficiency of EpoR under the expression of fluorescent protein and Cre recombinase;in addition,the use of self-cleaving peptide P2A avoids the influence of fusion proteins on theirs function and subcellular localization.Statistics analysis on sex,number of litters and genotype of newborn mice,blood parameters and erythropoiesis related parameters of adult mice demonstrated that the EpoR was fully functional in the EpoR-tdTomato-Cre mice.Then,we conducted more in-depth experimental studies on the expression and function of EpoR.Erythroblastic islands(EBI),consisting of central macrophages and surrounded erythroid cells,are niches for erythropoiesis,and provide essential microenvironment for development of erythroid cells.The expression of EpoR in erythroid cells and EBI macrophages was detected in EpoR-tdTomato-Cre mice.First,we improved the flow cytometry analysis method of erythroid cells.Flow cytometry analyses showed that in erythroid cells,EpoR-tdTomato was expressed in erythroid cells at all stages,among which CFU-E and Pro stages were the highest,and then gradually decreased.In mouse fetal liver,about 36%of macrophages expressed EpoR,and in mouse bone marrow the proportion was about 6.5%.Imaging flow cytometry analyses showed that in mouse fetal liver and bone marrow,more than 90%of EBI macrophages expressed EpoR,and the expression EpoR-tdTomato was readily detected in these surrounded erythroid cells.Another important function of EpoR-tdTomato-Cre mice was to use the Cre/Loxp system to achieve conditional gene edit in EpoR expressing cells.The efficiency of Cre recombinase in EpoR-tdTomato+erythroid cells and EBI macrophages of EpoRtdTomato-Cre+/+mice were at least 86%.These results showing that in EpoRtdTomato-Cre mice,the fluorescence of tdTomato could accurately reflect the expression level of EpoR,and conditional gene editing could be achieved in EpoR expressing cells.Accumulating evidence showed that EpoR might be expressed on cells besides erythroid cells.To apply EpoR-tdTomato-Cre mice in identifying EpoR expressing cells,we focused to check the expression of EpoR on macrophages which were related to the removal of damaged and senescent red blood cells and bone marrow hematopoietic cells,which was related with erythropoiesis.Spleen RPM and liver Kupffer cells were closely related to the removal of damaged and senescent red blood cells.Flow cytometry analyses showed that EpoR-tdTomato was expressed on about 7%of spleen macrophages and 23%of liver macrophages respectively.Analysis about the molecular surface markers revealed that these EpoR-tdTomato+macrophages were spleen RPM and liver Kupffer cells,respectively.To compare the functional differences of EpoR+cells from different tissues,we performed transcriptome analysis with the enriched mouse spleen,liver and BM EpoR+macrophages.GO analysis results showed that the signal pathways enriched by genes highly expressed in mouse spleen EpoRtdTomato+macrophages included cytokine production,inflammatory response,cellular iron homeostasis,and heme metabolism;The highly expressed genes from mouse liver EpoR-tdTomato+macrophages were mainly enriched in signal pathways such as inflammation response,cytokine production,and vasculature development;the signal pathways enriched by the highly expressed genes in mouse bone marrow EpoRtdTomato+ macrophages were related with cell proliferation and erythrocyte homeostasis.EpoR was also expressed in subtypes of hematopoietic stem and progenitor cells(HSPC),including hematopoietic stem cells(HSC),multipotent progenitor cells(MPP),myeloid progenitor cells(CMP),and megakaryocytic erythroid progenitor cells(MEP).Flow cytometry analyses revealed that EpoR positive proportions in these cells were about 98%,8.8%,20%,and 97%,respectively.In addition,about 33%of megakaryocytes expressed EpoR;and about 3%of B cells were EpoR+cells.Notably,hematopoiesis was significantly altered in the bone marrow of EPO-injected mice with a clear bias towards erythropoiesis.The results of in vivo Edu incorporation assay showed that EPO could selectively promote the proliferation of EpoR+cells and increase their proportion and number in mouse bone marrow.These results revealed the mechanism that EPO induced the bias of hematopoiesis to erythropoiesis.Our findings imply previously unanticipated broad roles of EPO/EpoR in hematopoiesis.The EpoR-tdTomato-Cre mouse model could facilitate future studies of EPO/EpoR biology beyond hematopoiesis.