Etv2 (Ets IntroductionBased on observations that several single-gene mutations severely affect both endothelial cell (EC) and hematopoietic cell (HPC) lineages, ECs and HPCs are proposed to originate from common precursors called hemangioblasts. 1,2 In zebrafish, cloche is the most classic mutant affecting both ECs and HPCs. 3,4 Deleting Flk-1, a major signaling receptor for VEGF, in mice causes defects in both ECs and HPCs. 5,6 However, in vitro differentiation of Flk-1-null embryonic stem cells (ESCs) generates both ECs and HPCs, albeit at much lower efficiencies. 7,8 These findings demonstrate that Flk-1 is dispensable for the differentiation of hemato-endothelial precursors but is required for the proliferation of these progenitor cells.Etv2 (Ets variant 2), identified in zebrafish as etsrp71, has been reported to be a key molecule for investigating In etsrp71 mutants, vasculogenesis and myelopoiesis are affected, leaving primitive erythropoiesis relatively intact. Conversely, overexpression of etsrp71 induces ectopic EC generation indicating a strong vasculogenic potential of etsrp71. 12 In mice, Etv-2 disruption results in a lack of Flk-1 expression, leading to the total loss of ECs and HPCs. 10,13 In ESCs, Etv2 overexpression up-regulates Flk-1 and subsequent EC generation. 10 From these results, Etv2 was hypothesized to be induced by bone morphogenic protein (BMP), Notch, and Wnt signaling and then transactivate Flk1. However, the phenotype discrepancy between the etsrp71 zebrafish mutant and Etv2-knockout mice raised the possibility that Etv2 may have more complex functions than proposed as an activator of Flk-1. Recent studies in zebrafish showed that, in addition to Flk-1, EC or HPC genes can also be up-regulated more robustly by Etv2. 14,15 Using Etv2 Venus knock-in mice and Etv2-null ESC lines (Etv2-Venus ϩ/Ϫ and Etv2 Ϫ/Ϫ , respectively), the role of Etv2 and downstream target genes in EC and HPC development from mesoderm were investigated in the present study. Methods Etv2-Venus ESCs and miceTargeting vector for the mouse Etv-2 locus was constructed using the Gateway cassette vector system containing Venus and Cre removable Neo resistance cassette. 16 Genomic sequences corresponding to 111658-119169 (accession number 167978.4) on the 5Јside and 119172-122639 (accession number 167978.4) on the 3Ј side were used as homology arms for recombination, and Venus was subcloned in-frame into the endogenous Etv2 allele. The resulting targeting construct and TT2 ESCs were used to generate Etv2-Venus knock-in mice (CDB0675K). Original and modified Etv2 loci are depicted in supplemental Figure 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Etv2 locus on the other allele was targeted to generate homozygous Etv2-null ESCs. These Etv2-Venus mice or gene-trap line 141H7 were used to generate Etv2-null embryos. 13 RNA isolation and expression profilingRNA was purified using the RNeasy MinElute column (QIAGEN) and reverse transcribed by Revatrace (TOYOBO). ...

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Background: Early mesoderm can be classified into Flk-1+ or PDGF receptor alpha (PDGFRα)+ population, grossly representing lateral and paraxial mesoderm, respectively. It has been demonstrated that all endothelial (EC) and hematopoietic (HPC) cells are derived from Flk-1+ cells. Although PDGFRα+ cells give rise to ECs/HPCs in in vitro ES differentiation, whether PDGFRα+ population can become hemato-endothelial lineages has not been proved in mouse embryos. Results: Using PDGFRαMerCreMer mice, PDGFRα+ early mesoderm was shown to contribute to endothelial cells including hemogenic ECs, fetal liver B lymphocytes, and Lin-Kit+Sca-1+ (KSL) cells. Contribution of PDGFRα+ mesoderm into ECs and HPCs was limited until E8.5, indicating that PDGFRα+/Flk-1+ population that exists until E8.5 may be the source for hemato-endothelial lineages from PDGFRα+ population. The functional significance of PDGFRα+ mesoderm in vascular development and hematopoiesis was confirmed by genetic deletion of Etv2 or restoration of Runx1 in PDGFRα+ cells. Etv2 deletion and Runx1 restoration in PDGFRα+ cells resulted in abnormal vascular remodeling and rescue of fetal liver CD45+ and Lin-Kit+Sca-1+ (KSL) cells, respectively. Conclusions: Endothelial and hematopoietic cells can be derived from PDGFRα+ early mesoderm in mice. PDGFRα+ mesoderm is functionally significant in vascular development and hematopoiesis from phenotype analysis of genetically modified embryos. Developmental Dynamics 242:254–268, 2013. © 2013 Wiley Periodicals, Inc.Key FindingsPDGF receptor alpha–positive mesoderm contributes to endothelial and hematopoietic cells in physiological mouse embryogenesis.PDGF receptor alpha–positive mesoderm in early embryo is distinct from yolk sac blood island mesoderm, representing a source of hematopoietic cells on the embryo proper side.Genetic manipulation of Etv2 or Runx1 in PDGF receptor alpha–positive mesoderm demonstrates the functional significance of this mesoderm subset in vascular development and hematopoiesis.