PDGF Receptor Alpha+ Mesoderm Contributes to Endothelial and Hematopoietic Cells in Mice

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 Findings PDGF 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. Keywords: PDGFRα, Flk-1, Runx1, endothelial, hematopoiesis INTRODUCTION Mesoderm is an intermediate state for epiblasts or ES cells to develop into endothelial (EC) and hematopietic cells (HPC). Early mesoderm can be classified grossly into Flk-1+ lateral-extraembryonic or PDGFRα+ paraxial mesoderm. While Flk-1+ mesoderm has been shown to contribute to virtually all HPCs and ECs (Lugus et al., 2009), several reports indicate that HPCs and ECs originate from somite, intraembryonic mesenchyme, or allantois containing PDGFRα+ mesoderm (Jukkola et al., 2005; Bertrand et al., 2005; Zeigler et al., 2006; Corbel et al., 2007). Among candidate origins for HPCs and hematopoietic stem cells (HSCs), yolk sac (YS) (Kataoka et al., 1997; Yoder et al., 1997; Samokhvalov et al., 2007) and lateral mesoderm (Wasteson et al., 2008) consist mainly of Flk-1+ cells with few or not so many PDGFRα+ cells. In contrast, more PDGFRα+ cells are present in intraembryonic para-aortic splanchnopleura (p-Sp) (Cumano et al., 1996), placenta (Gekas et al., 2005), and allantois, raising the possibility that PDGFRα+ cells can generate HPCs from these sites (Takakura et al., 1997). It has been demonstrated that PDGFRα+ mesoderm generates ECs and HPCs in ES differentiation culture (Sakurai et al., 2006). However, in vitro culture system defines each cell population by marker staining but lacks anatomical information that is critical to understand the physiological differentiation process. In mouse embryos, we have demonstrated that Flk-1+/PDGFRα+ cells accumulate in the absence of Etv2, failing to differentiate into Flk-1+/PDGFRα-cells (Kataoka et al., 2011). This suggests that PDGFRα+ cells can contribute to ECs and HPCs in mouse embryogenesis. In mouse development, however, how PDGFRα+ population including Flk-1+/PDGFRα+ cells contribute to various cell types has not been thoroughly evaluated. It is also important to confirm if the differentiation pathway in in vitro ES cell differentiation can be recapitulated in the real animal. In ES differentiation, it is expected that PDGFRα+/Flk-1+ cells are multi-potential for hemato-endothelial, muscle, or mesenchymal lineages partly due to the greater plasticity of differentiating ES cells. Since Flk-1+ cells have been shown to differentiate into skeletal muscle and cardiomyocytes in mouse embryos (Motoike et al., 2003), it is possible that PDGFRα induction in Flk-1+ cells might enforce the differentiation of Flk-1+ cells preferentially into muscle or mesenchymal lineages in the in vivo context. Therefore, we examined if PDGFRα+ cells contribute to ECs and HPCs in mouse embryos where differentiation is controlled in a more physiological manner. For this purpose, PDGFRα-MerCreMer (PRα-MCM) knock-in mice, expressing tamoxifen (Tmx) inducible MerCreMer (MCM) under control of the PDGFRα locus (Fig. 1A), was crossed with ROSA26-LacZ or YFP reporter strains (PRα-MCM-LacZ or PRα-MCM-YFP mice) to trace labeled PDGFRα+ cells in mouse embryos. We focused on ECs and HPCs derived from PDGFRα+ cells, as this may help to clarify the origin of HSCs that are one of the most important cell types to be created for therapeutic purposes. Fig. 1 A: Generation of PDGFRα-MerCreMer (PRα-MCM) knock-in mice. Tmx-inducible MerCreMer was knocked into the PDGFRα locus using homology arms corresponding to 5′ side, 79,307–85,194; 3′ side, 85,253–89,284 ... RESULTS PDGFRα Mesoderm Is Distinct From Extraembryonic Runx1+ Mesoderm in Early Embryos To locate the PDGFRα+ mesoderm, E7.5 neural plate (Fig. 1B), E8.0 head fold (Fig. 1C), or E8.5 somite stage (Fig. 1D), embryos were immunostained by PDGFRα, Flk-1, and Runx1 antibodies. As we reported, PDGFRα and Flk-1 stained almost distinct subset of mesoderm with some overlap in lateral mesoderm closer to the paraxial region (Kataoka et al., 1997, 2011). Runx1 was used to stain HPC precursors including erythroid progenitors and part of HSCs (Tanaka et al., 2012). No clear overlap was observed between PDGFRα+ and Runx1+ mesoderm, indicating that PDGFRα or Runx1 specifies distinct mesoderm population. This result was also confirmed by FACS analysis of NP- and HF-stage Runx1-Venus Knock-in embryos, in which almost no PDGFRα+/Runx1+ cells were detected (Fig. 1E). In situ hybridization for Runx1 also revealed that its expression is limited in the proximal region of the extraembryonic yolk sac, namely the blood island, validating that our immunostaining by Runx1 antibody for multi-color detection correctly reflects Runx1 in situ hybridization (data not shown). These findings suggest that any HPCs coming from PDGFRα+ cells develop from those cells that do not express Runx1 in these stages. At E7.5–8.5, we were able to detect an area stained by both PDGFRα and Flk-1. This double-positive population almost disappeared at E9.5 (see Fig. 6C), indicating that vasculogenic capacity in PDGFRα+ cells is dependent on Flk-1 and is limited in early time frame during embryogenesis. Fig. 6 A: PDGFRα+ cells labeled at E8.5 contribute to fetal liver HPCs, but with lower efficiency. By E9.5 labeling, almost no PDGFRα+ cells contribute to fetal liver HPCs. a: After E8.5 Tmx injection, fetal liver HPCs were analyzed. YFP+-labeled ... PDGFRα+ Cells Labeled at E7.5–8.0 Contribute to Endothelial Cells Including Aorta-Gonad-Mesonephron (AGM) To trace the fate of early mesoderm PDGFRα+ cells, pregnant females were injected with Tmx at E7.5 or E8.0 and PRα-MCM-LacZ embryos were analyzed at E10.5. In PRα-MCM-LacZ embryos, cells labeled at E7.5–E8.0 distributed broadly inside the embryo including somite, head mesenchyme, and heart at E10.5. LacZ+ cells mainly distributed throughout the embryo proper side with fewer labeled cells in YS, indicating that PDGFRα+ early mesoderm almost exclusively contribute to the embryo proper (Fig. 2A). Histological analysis of LacZ-stained embryos revealed that PDGFRα+ cells labeled at E7.5 or E8.0 contribute widely to somites, mesenchyme, cardiomyocytes, and ECs (Fig. 2B). Fig. 2 A: Whole mount LacZ staining of PRα-MCM-LacZ embryos. To activate MerCreMer, Tmx was injected into pregnant females at E7.5 (neural plate stage, top panels) or E8.0 (head fold stage, middle panels), when PDGFRα starts to be clearly detected ... As we aimed to analyze the contribution of PDGFRα+ cells into ECs and HPCs, LacZ-stained embryos were examined if there were any ECs labeled, especially those regarded as hemogenic ECs. We found that some ECs were LacZ positive in PRα-MCM-LacZ embryos exposed to Tmx at E7.5–8.0 (Fig. 2B). Notably, ECs on the ventral side of aorta in the AGM region were LacZ positive, including HPCs budding from the EC layer (Fig. 2B). This finding demonstrates that PDGFRα+ cells labeled at E7.5–8.0 can contribute to hemogenic ECs that potentially give rise to definitive HPCs. Tmx injection at E6.5 failed to label PDGFRα+ mesoderm derivatives such as somites or paraxial mesoderm (Fig. 2C), which were extensively labeled by E7.5 injection. Rather, E6.5 Tmx injection labeled mainly extraembryonic part including YS HPCs, demonstrating the time frame specificity of labeling in PRα-MCM line (Fig. 2C), as 24 hr delay of Tmx injection resulted in the labeling of distinct populations. These findings suggest that PRα-MCM transgene has labeled in relevant cell types that are expected to be the descendants of PDGFRα+ cells including cardiomyocytes and head mesenchyme. Additionally, we observed LacZ-positive ECs including those with potential hemogenic capacity. Tmx injections at E6.5 and E7.5 also support our assumption that Tmx will be almost ineffective 24 hr after injection (Zovein et al., 2008), ensuring the time frame specificity of the PRα-MCM transgene.