HSCs are the founder cells of the adult hematopoietic system, and thus knowledge of the molecular program directing their generation during development is important for regenerative hematopoietic strategies. Runx1 is a pivotal transcription factor required for HSC generation in the vascular regions of the mouse conceptus - the aorta, vitelline and umbilical arteries, yolk sac and placenta 1, 2. It is thought that HSCs emerge from vascular endothelial cells through the formation of intra-arterial clusters 3 and that Runx1 functions during the transition from ‘hemogenic endothelium’ to HSCs 4, 5. Here we show by conditional deletion that Runx1 activity in vascular endothelial cadherin (VEC) positive endothelial cells is indeed essential for intra-arterial cluster, hematopoietic progenitor, and HSC formation. In contrast, Runx1 is not required in cells expressing Vav, one of the first pan-hematopoietic genes expressed in HSCs. Collectively these data show that Runx1 function is essential in endothelial cells for hematopoietic progenitor and HSC formation from the vasculature, but its requirement ends once or before Vav is expressed.

The chorio-allantoic placenta forms through the fusion of the allantois (progenitor tissue of the umbilical cord), with the chorionic plate. The murine placenta contains high levels of hematopoietic stem cells, and is therefore a stem cell niche. However, it is not known whether the placenta is a site of hematopoietic cell emergence, or whether hematopoietic cells originate from other sites in the conceptus and then colonize the placenta. Here, we show that the allantois and chorion, isolated prior to the establishment of circulation, have the potential to give rise to myeloid and definitive erythroid cells following explant culture. We further show that the hematopoietic potential of the allantois and chorion does not require their union, indicating that it is an intrinsic property of these tissues. These results suggest that the placenta is not only a niche for, but also a source of, hematopoietic cells.

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In mammals, the production of red blood cells is tightly regulated by the growth factor erythropoietin (EPO). Mice lacking a functional Epo gene are embryonic lethal, and studying erythropoiesis in EPO-deficient adult animals has therefore been limited. In order to obtain a preclinical model for an EPO-deficient anemia, we developed a mouse in which Epo can be silenced by Cre recombinase. After induction of Cre activity, Epo(KO/flox) mice experience a significant reduction of serum EPO levels and consequently develop a chronic, normocytic and normochromic anemia. Furthermore, compared with wild-type mice, Epo expression in Epo(KO/flox) mice is dramatically reduced in the kidney, and expression of a well-known target gene of EPO signaling, Bcl2l1, is reduced in the bone marrow. These observations are similar to the clinical display of anemia in patients with chronic kidney disease. In addition, during stress-induced erythropoiesis these mice display the same recovery rate as their heterozygous counterparts. Taken together, these results demonstrate that this model can serve as a valuable preclinical model for the anemia of EPO deficiency, as well as a tool for the study of stress-induced erythropoiesis during limiting conditions of EPO.

The chorio-allantoic placenta provides for the exchange of nutrients, gas, and waste between the fetus and mother. The murine placenta is also enriched for long term repopulating hematopoietic stem cells, and is therefore a stem cell niche (Gekas et al. Dev. Cell8, 365, 2005 and Ottersbach et al. Dev. Cell8, 377, 2005). However, it is not known if the placenta is colonized by hematopoietic stem cells that originate from other sites in the conceptus, or whether the placenta itself is also a site of hematopoietic cell emergence. The placenta is formed through the fusion of the allantois, a mesodermal extension from the primitive streak, with the chorionic plate, which is comprised of extraembryonic ectoderm and mesoderm. Here we show that the allantois and chorion, isolated before chorio-allantoic fusion and prior to the establishment of circulation, contain hematopoietic potential. Both allantois and chorion explants produced CD45 positive cells, some of which also expressed Mac-1 and Gr-1, when cultured on OP9 stromal cells in the presence of cytokines that promote myeloid cell differentiation. Cultures of allantois and chorion explants in the presence of lymphoid promoting cytokines induced B220 and CD19 positive B cells. Spatial and temporal analysis of Runx1, a marker for definitive hematopoiesis, showed Runx1 is not expressed in either the allantois or the chorion prior to fusion, but is expressed at the base of the allantois and at the chorio-allantoic junction following fusion. Both allantoic explant cultures, and cultures of whole embryos from which allantoises were removed in order to prohibit chorio-allantoic fusion showed that Runx1 expression in neither tissue is dependent upon fusion. We conclude that the allantois and chorion have intrinsic hematopoietic potential prior to the establishment of circulation. Our results furthermore suggest that the placenta is both a niche and a source of hematopoietic cells.