Erythropoiesis from acetyl LDL incorporating endothelial cells at the preliver stage

Abstract

Erythropoiesis is characterized by 2 waves of production during mouse embryogenesis: a primitive one originating from the yolk sac (YS) and a definitive one produced from both the YS and the embryo proper. How the latter wave is generated remains unclear. To investigate our hypothesis that endothelial cells (ECs) could generate erythroid cells, we designed a method to label ECs at 10 days after coitus. This labeling method associates 2 techniques: an intracardiac inoculation that allows molecules to be delivered into the bloodstream followed by a whole-embryo culture period. DiI-conjugated acetylated low-density lipoproteins (Ac-LDL-DiI) were used to specifically tag ECs from the inside. One hour after inoculation, DiI staining was found along the entire endothelial tree. Fluorescenceactivated cell sorter (FACS) analysis revealed that DiI ؉ cells were CD31 ؉ , CD34 ؉ , and CD45 ؊ , an antigen makeup characteristic of the endothelial lineage. Twelve hours after inoculation, 43% of DiI ؉ circulating cells belonged to the erythroid lineage. These cells expressed Ter119 and displayed an adult globin chain arrangement; thus they belonged to the definitive lineage as confirmed in erythroid colony formation. IntroductionDuring mouse embryogenesis, hematopoiesis begins in the extraembryonic yolk sac (YS) at 7.5 days after coitus (dpc), shifting to fetal liver at midgestation, then to spleen, and finally to bone marrow shortly before birth. Classical embryological experiments, clonal culture assays, and genetic studies in the mouse have demonstrated the existence of 2 distinct waves of hematopoietic emergence: a transient one, mainly restricted to erythropoiesis occurring in the YS blood islands prior to circulation between YS and embryo, and a definitive one originating from both the YS and the embryo proper. Consistent with pioneer studies on the avian model, the embryonic site has been identified in the aortic region-the so-called para-aortic splanchnopleura (p-Sp)/aortagonad-mesonephros (AGM) region-and was shown to be capable of producing hematopoietic cells (HCs) including hematopoietic stem cells (HSCs). 1,2 In addition, in both avian and mouse species, the YS was shown to be able to produce definitive HSCs, including cells of erythroid lineage, at the same time as or even slighly before the AGM formation. [3][4][5][6][7][8] Primitive and definitive erythropoiesis yields mature erythrocytes distinguishable by their morphology and the hemoglobin types they express. Mature primitive erythrocytes are nucleated cells containing embryonic as well as adult hemoglobins, whereas mature definitive erythrocytes are smaller enucleated red blood cells committed to adult hemoglobin synthesis. [9][10][11] In the YS as well as in the aorta, HCs and ECs develop in close association. This proximity has prompted earlier embryologists to assume the existence of a putative ancestral mesodermal progenitor for ECs and HCs, the so-called hemangioblast. 12 In addition to the shared expression of several markers, gene targeting experimen...

The origin and fate of yolk sac hematopoiesis: application of chimera analyses to developmental studies

“…Hemogenic endothelial cells have been shown to arise in cultures of in differentiating ES cell cultures (Nishikawa et al, 1998). Further support or a connection between endothelial and hematopoietic differentiation was provided by a study using dye (Ac-LDL-DiI) labeling of endothelial cells at E10, with subsequent generation of Dil positive erythrocytes, indicating that they were potentially endothelial cell-derived (Sugiyama et al, 2003). One study difficult to reconcile with this emerging view or intersecting endothelial and hematopoietic development was performed by Bertland, who identified markers for hematopoietic progenitors in the AGM region that possess activity of long-term reconstitution of sublethally irradiated mice.…”

Tracing the hemangioblast during embryogenesis: developmental relationships between endothelial and hematopoietic cells

“…These results demonstrate, for the first time in the mouse embryo, the generation of hematopoietic cells from an endothelial intermediate, using an in vivo tracing. We thus provide evidence for the release of these cells into the circulation and put forward the hypothesis that they are able to colonize the fetal liver and generate definitive erythrocytes in vivo (Sugiyama et al, 2003).…”

Regulation of hematopoietic cell clusters in the placental niche through SCF/Kit signaling in embryonic mouse

“…For this procedure, 0.2-0.5 l of anti-Kit neutralizing antibody (ACK2) (eBioscience) and purified rat IgG2b (isotype control; eBioscience) were administered to ICR mouse embryos at 10.25 dpc, a stage at which we can cut the avascular area of yolk sac with minimal bleeding and inject materials into the heart, by intra-cardiac injection, as previously described (Kulkeaw et al, 2009;Sugiyama et al, 2003). Briefly, embryos in PBS were visualized under a stereomicroscope (Leica Microsystems MZ6, Wetzlar, Germany).…”

“…Injected embryos were transferred to culture bottles containing rat serum supplemented with 2 mg/ml glucose in a WEC system (Ikemoto Scientific Technology, Tokyo, Japan) and cultured for 6 hours in the case of intracardiac injection or 42 hours in the case of CM-DiI labeling at 37°C in the dark with a continuous supply of gas (60% O 2 and 5% CO 2 balanced with N 2 ) (Kulkeaw et al, 2009;Osumi-Yamashita et al, 1997;Sugiyama et al, 2003;Sugiyama et al, 2005;Sugiyama et al, 2007). After WEC, embryos exhibiting no conspicuous bleeding or anomalies were analyzed.…”