ABSTRACT
It is now well-established that hematopoietic stem cells (HSCs) and progenitor cells originate from a specialized subset of endothelium, termed hemogenic endothelium (HE), via an endothelial-to-hematopoietic transition. However, the molecular mechanisms determining which endothelial progenitors possess this hemogenic potential are currently unknown. Here, we investigated the changes in hemogenic potential in endothelial progenitors at the early stages of embryonic development. Using an ETV2::GFP reporter mouse to isolate emerging endothelial progenitors, we observed a dramatic decrease in hemogenic potential between embryonic day (E)7.5 and E8.5. At the molecular level, Runx1 is expressed at much lower levels in E8.5 intra-embryonic progenitors, while Bmi1 expression is increased. Remarkably, the ectopic expression of Runx1 in these progenitors fully restores their hemogenic potential, as does the suppression of BMI1 function. Altogether, our data demonstrate that hemogenic competency in recently specified endothelial progenitors is restrained through the active silencing of Runx1 expression.
Subject(s)
Core Binding Factor Alpha 2 Subunit/metabolism , Embryonic Development , Endothelial Progenitor Cells/metabolism , Gene Silencing , Hemangioblasts/cytology , Animals , Bone Morphogenetic Protein 4/metabolism , Embryo, Mammalian/metabolism , Embryonic Development/genetics , Endothelial Progenitor Cells/cytology , Female , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/metabolism , Hemangioblasts/metabolism , Hematopoiesis/genetics , Immunophenotyping , Male , Mice, Inbred ICR , Oligonucleotide Array Sequence Analysis , Polycomb Repressive Complex 1/antagonists & inhibitors , Polycomb Repressive Complex 1/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Single-Cell Analysis , Smad Proteins/metabolism , Transcription Factors/metabolismABSTRACT
BACKGROUND: ETV2 has been identified as an important player in embryonic hematopoiesis. However, the cell populations in which this transcription factor is expressed and operates during blood specification remain to be fully characterized. Here we address these issues using ES cells and a transgenic mouse line expressing green fluorescent protein (GFP) under the control of ETV2 regulatory elements, allowing us to observe the tight association between ETV2 expression and the initiation of hematopoiesis. RESULTS: Both in differentiating ES cells and gastrulating embryos ETV2::GFP is mostly found co-expressed with endothelial markers and defines a subset of cells with greatly enriched primitive erythroid potential. Upon culture ETV2::GFP cells rapidly up-regulate CD41, down-regulate endothelium cell surface markers and generate definitive hematopoietic progenitors. Altogether these characteristics represent the hallmark of hemogenic endothelium cells, a specialized endothelium originating from the hemangioblast and giving rise to hematopoietic cells. Importantly, ETV2 deficiency results in a complete absence of hemogenic endothelium in differentiating ES cells and gastrulating embryos. CONCLUSIONS: Altogether our data reveal that ETV2 marks hemogenic endothelium in gastrulating embryos and is absolutely required for the formation of this precursor at the onset of hematopoiesis. These results enhance our understanding of embryonic hematopoiesis and the factors driving hemogenic endothelium specification.
Subject(s)
Endothelium, Vascular/embryology , Hemangioblasts/metabolism , Hematopoietic Stem Cells/metabolism , Transcription Factors/genetics , Animals , Biomarkers/metabolism , Blood/metabolism , Embryo, Mammalian , Endothelium, Vascular/metabolism , Gastrulation/genetics , Gene Expression Regulation, Developmental/physiology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hemangioblasts/physiology , Hematopoiesis/genetics , Hematopoiesis/physiology , Hematopoietic Stem Cells/physiology , Mice , Mice, Transgenic , Neovascularization, Physiologic/genetics , Transcription Factors/metabolism , Transcription Factors/physiology , Transgenes , Validation Studies as TopicABSTRACT
During embryonic development, the emergence of hematopoiesis and vasculogenesis is tightly associated, with many transcription factors implicated in both developmental processes. Among those factors, ETV2 acts at the top of the hierarchy and controls the formation of both lineages. However, it is not known at which stage of mesoderm development ETV2 is acting and whether ETV2 activity is further required once mesodermal precursors have been specified to the hematopoietic and endothelial fates. In this study, we characterize the developmental window during which ETV2 expression is required for hematopoietic and endothelial development. Using cre-mediated deletion of ETV2, we demonstrate that ETV2 is acting prior to or at the time of FLK1 expression in mesodermal precursors to initiate the hematopoietic and endothelial program. Using the in vitro differentiation of embryonic stem cells as a model system, we further show that ETV2 re-expression in Etv2(-/-) Flk1-negative precursors drives hematopoiesis specification and switches on the expression of most genes known to be implicated in hematopoietic and endothelial development. Among the downstream targets of ETV2, we identify the transcription factors SCL, GATA2, and FLI1 known to operate a recursive loop controlling hematopoietic development. Surprisingly, SCL re-expression in Etv2(-/-) cells fully rescues hematopoiesis, while the re-expression of FLI1 or GATA2 promotes only a very limited rescue. Altogether, our data establish that ETV2 is required very transiently to specify mesodermal precursors to hematopoiesis and vasculogenesis and that SCL is one of the key downstream targets of ETV2 in controlling hematopoietic specification.
