Temporal-Spatial Mapping Of Hematopoietic Progenitors In The Embryo Reveals a Differentially Regulated Program Of Endothelial-To-Hematopoietic Transition In The Yolk Sac

It is established that hemogenic endothelium (HE) in major embryonic arteries is the source of hematopoietic stem cells (HSC), and the Runx1-mediated endothelial-to-hematopoietic transition in these sites depends both on arterial specification and on the establishment of blood flow. However, two waves of hematopoietic progenitors emerge prior to HSC specification in the mammalian embryo, yielding primitive and erythro-myeloid(EMP)-definitive hematopoiesis. Each has distinct lineage potential and temporal regulation, suggesting that each wave has a required, non-redundant function. Indeed, both are required for embryonic survival (Fujiwara et al., PNAS, 1996; Chen et al., Cell Stem Cell, 2011), and can sustain hematopoiesis in the murine embryo until birth in the absence of functional HSC. Commencing with the onset of cardiac function at embryonic day (E)8.25, EMP emerge in a Runx1-dependent manner contemporaneous with vasculogenesis and angiogenesis in the yolk sac. Moreover, EMP emergence temporally overlaps both with primitive hematopoietic emergence and HSC specification in the adjacent embryonic arteries (Palis et al., Development, 1999). Given this temporal overlap, we reasoned that identifying the spatial localization and cellular origin of EMP emergence would provide important insight into their regulation and function.

We purified the temporally overlapping hematopoietic progenitor populations in the yolk sac, and confirmed their lineage identity by in vitro culture. Although previous studies associated CD41^hi cells with emerging EMP in the E8 blood islands (Ferkowicz et al., Development, 2003; Li et al., Stem Cells Dev., 2005), we now demonstrate that CD41^hi cells at E9 represent maturing Gp1bβ+ megakaryocytes, while Kit^hi EMP remain CD41^Mid. Furthermore, both CD45 and CD16/32 are co-expressed on primitive macrophages and EMP. These findings suggest kit is the most precise single marker to identify emerging EMP in the yolk sac. Notably, despite the loss of Kit^hiCD41+CD16/32+ EMP in Runx1-/- yolk sacs, specification of single-positive CD41^hi and CD16/32+ cells is intact, confirming the developmental origin of the megakaryocyte and macrophage lineages from primitive hematopoietic progenitors.

As EMP and primitive hematopoietic emergence temporally overlap but differ in lineage potential, we expected these populations to arise within spatially distinct regions of the yolk sac. Surprisingly, we detect kit+ EMP at E8.25 in the proximal yolk sac, both within and adjacent to the blood islands, where primitive erythroid cells emerge. Many Kit+ cells co-express Runx1 and CD31, have a polygonal, endothelial morphology, and appear integrated into the vascular network, consistent with the hemogenic endothelial origin of EMP. The close association of polygonal and round Kit+ cells further support EMP emergence via an endothelial-to-hematopoietic transition. Prior to E9.5, clusters of Kit+Runx1+ EMP remain in the proximal yolk sac, despite the broad distribution of primitive erythroid cells throughout the yolk sac vasculature following the onset of circulation. This is consistent with our observation that EMP clusters are associated with the vessel walls and may be unable to freely circulate. Between E9-10, the spatial distribution of HE broadens, as both small and large clusters of EMP form more distally in the yolk sac. However, unlike HSC, EMP emerge in both small and large vessels. To address the influence of arterial specification and blood flow on EMP emergence, we analyzed yolk sacs from Ncx1-/- mouse embryos, and found normal cluster morphology and spatial distribution, as well as numbers of immunophenotypic EMP, despite the lack of circulation and vascular remodeling. We next asked whether emerging EMP are preferentially associated with arterial vessels, which are predetermined to arise in the posterior region of the yolk sac (Wang et al., Cell, 1998). However, no anterior-posterior asymmetry of EMP clusters was evident in wildtype or Ncx1-/- yolk sacs. We conclude that EMP emergence from HE is initially restricted to the proximal region of the yolk sac that also generates primitive hematopoiesis, but subsequently extends throughout the yolk sac in both arterial and venous vasculature. Our results highlight that distinct populations of HE exist in the conceptus, and are subject to diverse regulatory mechanisms, which may control progenitor versus stem cell fate.