| Hematopoietic stem cells are the main source of mature blood cells including myeloid cells and lymphocytes.During mouse embryonic development,hematopoietic stem cells mainly originated from the aorta-gonad-mesonephros(AGM)region on the embryonic day 10.5(E10.5),when the aortic endothelium produced hematopoietic cells through endothelial to hematopoietic transition.During this process,flat-shaped hemogenic endothelia transform into round-shaped hematopoietic cells,and gathered to form hematpoietic clusters by budding.The clusters contain precursors of hematopoietic stem cell,which eventually develop into mature hematopoietic stem cells and migrate to the fetal liver for expansion,and then colonized to bone marrow.During the development of embryonic hematopoietic stem cells,it has been impossible to accurately capture hematopoietic stem cells and their related populations due to the lack of specific surface marker.Combined with single-cell induction transplantation system,we obtained high-specific surface markers and enriched hematopoietic stem cell precursors efficiently at single-cell level for the first time in our previous studies.Also combining with single-cell transcriptome sequencing,we systematically analyzed the dynamic molecular expression characteristics of single cells in the whole process of hematopoietic stem cell development.In this paper,the data of single cell transcriptome in the whole process of hematopoietic stem cells development were further explored,and the expression characteristics of molecules in early embryonic endothelial cells,hematopoietic stem cell precursors,fetal liver and adult hematopoietic stem cells populations were analyzed,and we obtain a set of genes whose expression is up-regulated during hematopoietic stem cell specialization,and the Hlf(hepatic leukemia factor)gene gradually up-regulated and significantly highly expressed in hematopoietic stem cells from hematopoietic stem cell precursors to hematopoietic stem cells was screened out.Recent studies have shown that Hlf is specifically expressed in fetal liver and adult hematopoietic stem cells,suggesting that Hlf may also specifically mark hematopoietic stem cell precursors and hematopoietic stem cells in early embryo.Therefore,we constructed Hlf-tdTomato fluorescent reporter mice and Hlf-CrexER lineage tracing mice.The expression profile of newly constructed Hlf-tdTomato fluorescent reporter mice was identified,and the results of histochemical staining showed that the hematopoietic population co-expressing RUNX1 and CD31 expressed Hlf-tdTomato in the AGM on E10.5,while the CD31+endothelial cells did not express Hlf-tdTomato.The flow cytometry results also showed that Hlf-tdTomato was not expressed in the endothelial cell population(CD31+CD41-CD43-CD45-)in AGM on E10.5,but more than 40%of cells in hematopoietic cluster(CD31+Kit+)expressed Hlf-tdtomato.Nearly 40%of CD45-hematopoietic stem cell precursors and almost all CD45+hematopoietic stem cell precursors expressed Hlf-tdTomato in AGM region on E11.Furthermore,we found that only Hlf-tdTomato+ subsets showed competence of myeloid and lymphoid reconstruction 3 to 4 weeks after induction/transplantation in CD31+Kit+CD45+population on embryonic day 10 while Hlf-tdTomato+and Hlf-tdTomato-subsets in CD31+Kit+CD45-population showed this competence.In addition,the in vitro incubation of hematopoietic cluster cells(CD31+Kit+)on E11 showed that only Hlf+cells could produce hematopoietic stem cell phenotype-like cells.The above functional experiments show that the expression of Hlf can be used to specifically enrich functional hematopoietic stem cell precursors whith CD45+populaton.Then,the newly constructed Hlf-CrexER was hybridized with ROSA26 reporter mice,and the cell population expressing Hlf-CrexER was induced at different time points of early embryo development,so as to explore the dynamic changes of the contribution of early embryo Hlf-CrexER positive cells to each hematopoietic stem progenitor cell population and adult peripheral blood.The analysis of fetal liver hematopoietic stem cells by using surface marker combinations of Lin-Sca-1+Mac-11lowCD201+(LSM201),CD45+CD201+CD48-CD150+(ESLAM)and Lin-Sca-1+Kit+CD48-CD150+(LSKSLAM)showed that fetal liver was basically unlabeled after induction on E8.5.However,after induction on E9.5,E10.5,E11.5 and E13.5,the labeling efficiency of fetal liver hematopoietic stem cells increased gradually,especially after induction on E9.5 and E10.5,the average labeling efficiency jumped.The continuous detection of labeling efficiency of each lineage in peripheral blood of adult mice born after induction showed that the average labeling efficiency of each lineage in peripheral blood after induction on E9.5 was about 2%,which was significantly lower than that after induction on E10.5(about 20%),indicating that hematopoietic stem cell precursors and hematopoietic stem cell populations in AGM region may be fully labeled with the gradual increase of Hlf expression intensity during this period.Because hematopoietic stem cell precursors and hematopoietic stem cell populations in AGM region originated from endothelial cells,we further obtained constitutive mice by mating Tie2-Dre with Hlf-CrexER,and then hybridized with ROSA26 reporter mice,which can limit Hlf labeling to early endothelial cells more specifically.Through the detection of fetal liver hematopoietic stem cells,we found that the labeling efficiency of constitutive mice in fetal liver hematopoietic stem cells was not as high as that in E13.5 hematopoietic stem cells inducible mice.By comparing the labeling efficiency of the peripheral blood lineages of postnatal mice,we found that the labeling efficiency of peripheral blood lineages of constitutive mice was also lower than that of induced mice.In summary,through single cell transcriptomic analysis,Hlf,a molecule specifically expressed in hematopoietic stem cell precursors,fetal liver hematopoietic stem cells and adult hematopoietic stem cells was screened out,and Hlf-tdTomato fluorescent reporter mice and Hlf-CrexER lineage tracing mice were constructed.We further labeled the precursors of early embryonic hematopoietic stem cells by fluorescent reporting mouse model,and revealed the hematopoietic fate of Hlf-expressing cells at different developmental time points and the dynamic changes of their contribution to fetal liver hematopoietic stem cells by lineage tracing model.Furthermore,the constructed mouse model can be used in combination with various mice model.The combination of lineage tracing mice and Confetti mice can be applied to study the heterogeneity of hematopoietic stem cells.Using fluorescent reporting mice can explore the location of hematopoietic stem cells in bone marrow microenvironment and the mechanism of interaction between hematopoietic stem cells and other molecules,which is of great significance to study the origin and application of hematopoietic stem cells. |
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