EMP Emergence from Hemogenic Endothelium in the Mammalian Yolk Sac Is Independent of Flow and Arterial Identity, but Is Regulated By Canonical Wnt Signaling

Hematopoietic stem cells (HSCs) emerge from arterial vessels of the mouse embryo through a Runx1-dependent process of endothelial-to-hematopoietic transition beginning at embryonic day 10.5 (E10.5). This arterial endothelial-to-hematopoietic transition is known to require embryonic circulation as well as beta-catenin signaling within the endothelial precursor, known as hemogenic endothelium. However, embryonic survival is dependent on the earlier emergence of a robust wave of yolk sac-derived definitive erythro-myeloid progenitors (EMPs), which have unilineage as well as multilineage potential, including high-proliferative potential colony forming cell (HPP-CFC) potential (Palis et al., PNAS, 2001). Like HSCs, EMP specification is dependent on Runx1, suggesting that they also emerge from a hemogenic endothelial precursor. However, the spatial localization of EMPs in the yolk sac and the mechanisms governing their emergence are not well understood. To visualize emerging EMPs in the yolk sac, we performed whole-mount immunohistochemistry for Kit, which we have demonstrated to contain nearly all EMP potential at E9.5. Kit+ cells coexpress Runx1 and CD31, and a subset have a polygonal/endothelial morphology, appear integrated into the vascular network, and are associated with rounded Kit+ cells in clusters, features consistent with an endothelial-to-hematopoietic transition. However, unlike HSCs, which emerge from major embryonic arteries, clusters of EMPs are located in larger and smaller caliber vessels in branches of both the arterial and venous vasculature, which is spatially organized within the yolk sac. To determine if EMP emergence from the vasculature is dependent on embryonic blood flow, which is required for HSC emergence, we analyzed the yolk sacs of Ncx1-null embryos, which fail to initiate heart contractions and subsequently lack embryonic circulation. Despite the lack of vascular remodeling in these circulation-deficient yolk sacs, Ncx1-null EMPs displayed normal cluster morphology, including both polygonal and rounded kit+ cells, indicating the endothelial-to-hematopoietic transition can occur without the mechanical influence of blood flow.

To address whether EMP formation is responsive to other developmental signals, we utilized a yolk sac explant culture to evaluate the propensity of hemogenic endothelial cells to commit to hematopoiesis ex vivo. Culture of intact E8.5 yolk sacs for 48 hours with the canonical Wnt ligand Wnt3a resulted in an increase in both day 6-7 colony forming cells and day 13-14 HPP-CFC when compared with control yolk sacs. Preliminary treatment with Dkk1 alone did not adversely affect colony-forming activity when compared with untreated yolk sacs, and potentiation of endogenous canonical Wnt signaling with HLY78 did not augment colony production, suggesting that low levels of endogenous Wnt ligands are produced ex vivo. Despite the positive effect of Wnt3a on whole yolk sacs, treatment of isolated E9.5 Kit+CD41+CD16/32+ EMPs with Wnt3a did not increase colony formation, suggesting that Wnt signaling augments progenitor production at, or prior to, the hemogenic endothelial stage. Preliminary results utilizing imaging flow cytometry demonstrated increased beta-catenin intensity within the nuclear region in E9.5 Kit+VE-Cadherin/AA4.1+ endothelium following Wnt3a treatment, suggesting that hemogenic endothelial cells in the yolk sac are Wnt responsive. Consistent with this finding, in vitro Wnt3a treatment on primary E8.5-9.5 VE-Cadherin/AA4.1+CD16/32- endothelial cells resulted in upregulation of the beta-catenin target gene Axin2. To address whether Wnt signaling is endogenously active in vivo, we analyzed E8.5-E9 yolk sacs of BAT-gal reporter mice (Maretto et al., PNAS, 2003), and visualized a subset of cells with endothelial morphology expressing LacZ. Taken together, these data support the concept that EMPs, like HSCs, emerge from hemogenic endothelium. Surprisingly, this earlier endothelial-to-hematopoietic transition in the yolk sac is not dependent on blood flow or an arterial identity. However, similar to HSC emergence, EMP emergence from hemogenic endothelium is positively regulated by canonical Wnt signaling. These data highlight the presence of spatially, temporally, and functionally heterogeneous populations of hemogenic endothelium in the mammalian conceptus.