Study On Flk1~+ Cells During Mouse Early Embryogenesis By Lineage Tracing

In the early development of mammalian embryogenesis,hematopoietic development and vasculargenesis have been the focus of biological research.However,since the circulatory system was established before the appearance of the first hematopoietic stem cell(HSC),the relationship between hematopoiesis and vascular development has not been elucidated.At present,the common viewpoints on the origin of hematopoietic development can be divided into two types.The former believes that hematopoietic and endothelial cells(ECs)originate from the common mesoderm precursor,hemangioblast;the latter consider hematopoietic cells are differentiated by functionally specialized hematopoietic endothelium.The differentiation of pluripotent stem cells has also been a core issues in biology.Flk1(fetal liver kinase 1),one of the receptors for vascular endothelial growth factor(VEGF),is also a hallmark of mesodermal progenitor cells.Flk1~+cells represent one of the earliest mesodermal cells,they are able to differentiate into multiple mesoderm lineages,and have been first known for the bi-directional differentiation potential for hematopoietic and vascular endothelial lineages.Studies have shown that all adult hematopoietic lineages have undergone the expression phase of Flk1,showing that Flk1is closely related to the development of hematopoietic and vascular lineages.Toshiyuki Motoike et al.used a genetic lineage tracing system to prove that Flk1~+progenitor cells also contribute to the muscle lineage.In addition,Gorden Keller et al.also identified Flkl~+cells as a progenitor of the cardiovascular lineage.Recently,the adult mouse mammary fat pad was used as a model to provide direct evidence that Procr~+endothelial cells can differentiate into neonatal pericytes in adult mice.We believe that Flk1~+progenitor cells may have more extensive differentiation potential during mammalian early embryogenesis.In this study,to understand the multilineage differentiation potential of the mesoderm-derived Flk1~+progenitors in different parts of the early mouse embryogenesis,and to explore whether Flk1~+progenitors also differentiate into pericytes and other mesenchymal lineage.Based on the Cre-LoxP system-based conditional knockout strategy,Flk1-Cre and ROSA26 reporter mice were used to perform our tracing studies.In the reporter mice,the fate of Flkl~+progenitors was traced with the YFP~+population.In addition,combined mesoderm marker Flk1,hematopoietic cells specific marker CD45,endothelial cells specific markers CD31,CD144 and Emcn(endomucin),and pericytes specific markers PDGFR?and NG2,using immunohistochemistry,immunofluorescence,and other histological methods and flow cytometric analysis to solve our concerns.In the first part of the study,the immunohistochemical results of different sections of E8.5-10.5 Flk1-Cre;ROSA26-EYFP embryos were shown,such as the dorsal aorta(DA),In multiple YFP~+groups such as limb buds and yolk sac(YS),blood cells,CD31~+Emcn~+typical endothelial cells distributed along the vessel wall,and non-endothelial mesenchymal-like cells were observed.It suggests that the Flk1~+population may have wider potential in addition to the differentiation potential of the hematopoietic,vascular and muscle lineages.In the second part of the study,combined with endothelial surface specific markers,the double-labeled immunofluorescence results confirmed in the dorsal aorta,fetal liver and other hematopoietic sites CD31~-mesenchymal-like cells do exist in the YFP~+population.Finally,triple-labeled immunofluorescence results that combined with endothelial and pericyte markers showed that this group of cells that are non-hematopoietic and non-endothelial around blood vessels are PDGFR?~+NG2~+pericytes.Flow cytometric analysis also confirmed that Flk1~+progenitors not only contributed to the hematopoietic lineage,the vascular endothelial lineage,but also contributed to this group of pericytes.These findings suggest that Flk1~+progenitors not only contribute to hematopoietic,endothelial,muscle and cardiovascular lineages,but also have the potential to differentiate into pericytes.Interestingly,in different sites of early embryogenesis of Flk1-Cre;ROSA26-EYFP,such as dorsal aortas and limb buds,we observed that a group of CD31~+CD140b~+mesenchymal-like cells in the YFP~+population.Previous studies have shown that the endothelial-mesenchymal transition(EndMT)effect has been observed under the physiological and pathological conditions,demonstrating the differentiation potential of endothelial cells and their plasticity in vivo and in vitro.It is speculated that the CD31~+CD140b~+population may be endothelial cells derived from Flk1~+progenitors that are undergoing EndMT and gradually lose endothelial markers,and also begin to express mesenchymal markers.Whether the double markers positive group were the pericytes population that we observed in immunohistochemistry and whether the endothelial cells derived from Flk1~+progenitors actually contributed to the pericytes after undergoing EndMT are not yet clear.Further researches are needed.