Summary Hematopoietic potential arises in mammalian embryos before adult-repopulating hematopoietic stem cells (HSCs). At E9.5, we show the first murine definitive erythro-myeloid progenitors (EMPs) have an immunophenotype distinct from primitive hematopoietic progenitors, maturing megakaryocytes and macrophages, and rare B cell potential. EMPs emerge in the yolk sac with erythroid and broad myeloid, but not lymphoid, potential. EMPs migrate to the fetal liver and rapidly differentiate including production of circulating neutrophils by E11.5. While the surface markers, transcription factors and lineage potential associated with EMPs overlap with those found in adult definitive hematopoiesis, they are present in unique combinations or proportions that result in a specialized definitive embryonic progenitor. Further, we find that ES cell -derived hematopoiesis recapitulates early yolk sac hematopoiesis, including primitive, EMP and rare B cell potential. EMPs do not have long term potential when transplanted in immunocompromised adults, but can provide transient adult-like RBC reconstitution.

Directed cell movement is integral to both embryogenesis and hematopoiesis. In the adult, the chemokine family of secreted proteins signals migration of hematopoietic cells through G-coupled chemokine receptors. We detected embryonic expression of chemokine receptor messages by RT-PCR with degenerate primers at embryonic day 7.5 (E7.5) or by RNase protection analyses of E8.5 and E12.5 tissues. In all samples, the message encoding CXCR4 was the predominate chemokine receptor detected, particularly at earlier times (E7.5 and E8.5). Other chemokine receptor messages (CCR1, CCR4, CCR5, CCR2, and CXCR2) were found in E12.5 tissues concordant temporally and spatially with definitive (adult-like) hematopoiesis. Expression of CXCR4 was compared with that of its only known ligand, stromal cell-derived factor-1 (SDF-1), by in situ hybridization. During organogenesis, these genes have dynamic and complementary expression patterns particularly in the developing neuronal, cardiac, vascular, hematopoietic, and craniofacial systems. Defects in the first four of these systems have been reported in CXCR4- and SDF-1-deficient mice. Our studies suggest new potential mechanisms for some of these defects as well as additional roles beyond the scope of the reported abnormalities. Earlier in development, expression of these genes correlates with migration during gastrulation. Migrating cells (mesoderm and definitive endoderm) contain CXCR4 message while embryonic ectoderm cells express SDF-1. Functional SDF-1 signaling in midgastrula cells as well as E12.5 hematopoietic progenitors was demonstrated by migration assays. Migration occurred with an optimum dose similar to that found for adult hematopoietic cells and was dependent on the presence of SDF-1 in a gradient. This work suggests roles for chemokine signaling in multiple embryogenic events.

In the adult, platelets are derived from unipotential megakaryocyte colony-forming cells (Meg-CFCs) that arise from bipotential megakaryocyte/erythroid progenitors (MEPs). To better define the developmental origin of the megakaryocyte lineage, several aspects of megakaryopoiesis, including progenitors, maturing megakaryocytes, and circulating platelets, were examined in the murine embryo. We found that a majority of hemangioblast precursors during early gastrulation contains megakaryocyte potential. Combining progenitor assays with immunohistochemical analysis, we identified 2 waves of MEPs in the yolk sac associated with the primitive and definitive erythroid lineages. Primitive MEPs emerge at E7.25 along with megakaryocyte and primitive erythroid progenitors, indicating that primitive hematopoiesis is bilineage in nature. Subsequently, definitive MEPs expand in the yolk sac with Meg-CFCs and definitive erythroid progenitors. The first GP1b␤-positive cells in the conceptus were identified in the yolk sac at E9.5, while large, highly reticulated platelets were detected in the embryonic bloodstream beginning at E10.5. At this time, the number of megakaryocyte progenitors begins to decline in the yolk sac and expand in the fetal liver. We conclude that the megakaryocyte lineage initially originates from hemangioblast precursors during early gastrulation and is closely associated both with primitive and with definitive erythroid lineages in the yolk sac prior to the transition of hematopoiesis to intraembryonic sites. IntroductionHematopoiesis in the mouse embryo initiates shortly after the onset of gastrulation in the blood islands of the extraembryonic yolk sac. 1 Here, the close spatial and temporal arrangement of the first hematopoietic and endothelial precursors has long suggested that both cell types originate from a common hemangioblast progenitor. 2 This hypothesis has gained support from studies of embryonic stem cells cultured in vitro 3-7 and more recently been confirmed by the identification of blast-colony-forming cells (BL-CFCs) in mouse embryos that possess endothelial, as well as primitive erythroid, definitive erythroid, and multiple myeloid lineage potential. 8 A transient pool of primitive erythroid progenitors (EryPCFCs) emerges between embryonic days 7.25 to 9.0 (E7.25-E9.0) and generates large, nucleated ␤H1-globin-expressing erythroid cells that eventually enucleate upon maturation. 9-11 Subsequently, a second, overlapping wave of definitive erythroid progenitors (burst-forming units-erythroid [BFU-Es]), similar to those found later in the fetal liver and adult bone marrow, emerges in the yolk sac accompanied temporally by mast cell, granulocyte, macrophage, and multipotential high proliferative potential colony forming cells (HPP-CFCs). 9,10,12,13 These findings have suggested that the definitive wave of yolk sac hematopoietic progenitors is multilineage, while the initial primitive wave is unilineage, being specifically confined to the primitive erythroid lineage. 14 In the adult bone marr...

Key Points• Comparative global gene expression analysis of primary murine primitive, fetal definitive, and adult definitive erythroid precursors.• Primitive erythroblasts contain and accumulate high ROS levels and uniquely express the H2O2 transporting aquaporins 3 and 8.Erythroid ontogeny is characterized by overlapping waves of primitive and definitive erythroid lineages that share many morphologic features during terminal maturation but have marked differences in cell size and globin expression. In the present study, we compared global gene expression in primitive, fetal definitive, and adult definitive erythroid cells at morphologically equivalent stages of maturation purified from embryonic, fetal, and adult mice. Surprisingly, most transcriptional complexity in erythroid precursors is already present by the proerythroblast stage. Transcript levels are markedly modulated during terminal erythroid maturation, but housekeeping genes are not preferentially lost. Although primitive and definitive erythroid lineages share a large set of nonhousekeeping genes, annotation of lineage-restricted genes shows that alternate gene usage occurs within shared functional categories, as exemplified by the selective expression of aquaporins 3 and 8 in primitive erythroblasts and aquaporins 1 and 9 in adult definitive erythroblasts. Consistent with the known functions of Aqp3 and Aqp8 as H 2 O 2 transporters, primitive, but not definitive, erythroblasts preferentially accumulate reactive oxygen species after exogenous H 2 O 2 exposure. We have created a user-friendly Web site (http:// www.cbil.upenn.edu/ErythronDB) to make these global expression data readily accessible and amenable to complex search strategies by the scientific community. (Blood. 2013;121(6):e5-e13) IntroductionRBCs constitute an estimated 1 in 4 cells in the body and are necessary for tissue oxygen delivery. In the adult, RBCs are produced primarily in the BM where lineage-committed progenitors give rise to morphologically identifiable precursors. Erythroid precursors physically associate with macrophages and undergo several maturational cell divisions characterized by a progressive decrease in cell size, nuclear condensation, hemoglobin accumulation, and loss of RNA content. 1 These physical changes have been used to classify erythroid precursors into proerythroblast, basophilic, polychromatophilic, and orthochromatic erythroblast maturational stages. In mammals, orthochromatic erythroblasts enucleate to form reticulocytes that ultimately enter the circulation and complete their maturation.Erythroid cells are a critical component of the cardiovascular network, which constitutes the first functional organ system in the mammalian embryo. 2 "Primitive" erythroid cells first emerge in yolk sac blood islands. 3 We previously determined that primitive erythroid cells originate from a transient wave of committed progenitors in the yolk sac and mature as a semisynchronous cohort in the bloodstream, undergoing morphologic changes similar to those observed in defini...

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To better understand the relationship between the embryonic hematopoietic and vascular systems, we investigated the establishment of circulation in mouse embryos by examining the redistribution of yolk sac-derived primitive erythroblasts and definitive hematopoietic progenitors. Our studies revealed that small numbers of erythroblasts first enter the embryo proper at 4 to 8 somite pairs (sp) (embryonic day 8.25 [E8.25]), concomitant with the proposed onset of cardiac function. Hours later (E8.5), most red cells remained in the yolk sac. Although the number of red cells expanded rapidly in the embryo proper, a steady state of approximately 40% red cells was not reached until 26 to 30 sp (E10). Additionally, erythroblasts were unevenly distributed within the embryo's vasculature before 35 sp. These data suggest that fully functional circulation is established after E10. This timing correlated with vascular remodeling, suggesting that vessel arborization, smooth muscle recruitment, or both are required. We also examined the distribution of committed hematopoietic progenitors during early embryogenesis. Before E8.0, all progenitors were found in the yolk sac. When normalized to circulating erythroblasts, there was a significant enrichment (20-to 5-fold) of progenitors in the yolk sac compared with the embryo proper from E9.5 to E10.5. These results indicated that the yolk sac vascular network remains a site of progenitor production and preferential adhesion even as the fetal liver becomes a hematopoietic organ. We conclude that a functional vascular system develops gradually and that specialized vascular-hematopoietic environments exist after circulation becomes fully established. IntroductionA functional circulatory system is an early requirement for survival and growth of the mammalian embryo and is the first organ system to develop in the embryo. 1 The circulatory system is composed of vascular, hematopoietic, and cardiac components, each formed from discrete regions of mesoderm. The first endothelial cells and blood cells are generated in yolk sac blood islands beginning at embryonic day 7 (E7.0) in the mouse. By E8.0, thousands of nucleated primitive red blood cells have formed within a vascular plexus in the yolk sac. [2][3][4] Concurrently, the aorta and the peristaltic beating heart tube form in the embryo proper. In the next 36 hours, there is a remarkable increase in complexity of vascular and hematopoietic systems. The vascular plexus remodels and expands into an arborized network of specialized arteries and veins. Primitive erythroblasts from the yolk sac continue to divide and mature, and a second wave of hematopoiesis creating definitive (adultlike) progenitors originates in the yolk sac. 5,6 Thus, the early vascular system is the hematopoietic environment for primitive and definitive lineages until the specialized stromal microenvironment of the fetal liver (beginning E10), and later the adult bone marrow, is available. However, the nature of any specific interactions between early embryonic hematopo...

Mammals have 2 distinct erythroid lineages. The primitive erythroid lineage originates in the yolk sac and generates a cohort of large erythroblasts that terminally differentiate in the bloodstream. The definitive erythroid lineage generates smaller enucleated erythrocytes that become the predominant cell in fetal and postnatal circulation. These lineages also have distinct globin expression patterns. Our studies in primary murine primitive erythroid cells indicate that ␤H1 is the predominant ␤-globin transcript in the early yolk sac. Thus, unlike the human, murine ␤-globin genes are not up-regulated in the order of their chromosomal arrangement. As primitive erythroblasts mature from proerythroblasts to reticulocytes, they undergo a ␤H1-to ⑀y-globin switch, up-regulate adult ␤1-and ␤2-globins, and down-regulate -globin. These changes in transcript levels correlate with changes in RNA polymerase II density at their promoters and transcribed regions. Furthermore, the ⑀y-and ␤H1-globin genes in primitive erythroblasts reside within a single large hyperacetylated domain. These data suggest that this "maturational" ␤H1-to ⑀y-globin switch is dynamically regulated at the transcriptional level. Globin switching during ontogeny is due not only to the sequential appearance of primitive and definitive lineages but also to changes in globin expression as primitive erythroblasts mature in the bloodstream. IntroductionThe first blood cells to circulate during mammalian embryogenesis consist of large primitive red cells. In the mouse embryo, primitive erythroid cells are generated from a transient wave of committed progenitors found in the yolk sac between embryonic day (E) 7.25 and E9.0 of gestation. 1,2 Primitive proerythroblasts begin to emerge from blood islands beginning at E8.25, and enter the newly forming bloodstream, 3 where they undergo terminal maturation. They transition as a semisynchronous cohort of cells from proerythroblasts to enucleated erythrocytes progressively undergoing a loss of proliferative capacity, a decrease in cell size, accumulation of hemoglobin, and nuclear condensation. [4][5][6] Ultimately, they enucleate by E16.5 to become primitive erythrocytes that can circulate for several days after birth. 7 After E11.5, primitive red cells are joined by increasing numbers of smaller definitive erythrocytes that are released from the fetal liver after enucleating. Over the ensuing 5 days, definitive erythrocytes become the predominant cell type in the fetal circulation and ultimately become the exclusive red-cell lineage in the adult.The sine qua non of red cells is their accumulation of large amounts of hemoglobin composed of globin tetramers encoded by the ␣-and ␤-globin gene loci. Examination of globin expression in circulating human and mouse blood cells indicated that 5Ј members of both globin loci are expressed in the embryo, while 3Ј members are expressed in the adult, leading to the concept of globin switching during ontogeny. 8 Since this switch in globin expression coincided temporally with t...

Adult-repopulating hematopoietic stem cells (HSCs) emerge in low numbers in the midgestation mouse embryo from a subset of arterial endothelium, through an endothelial-to-hematopoietic transition. HSC-producing arterial hemogenic endothelium relies on the establishment of embryonic blood flow and arterial identity, and requires β-catenin signaling. Specified prior to and during the formation of these initial HSCs are thousands of yolk sac-derived erythro-myeloid progenitors (EMPs). EMPs ensure embryonic survival prior to the establishment of a permanent hematopoietic system, and provide subsets of long-lived tissue macrophages. While an endothelial origin for these HSC-independent definitive progenitors is also accepted, the spatial location and temporal output of yolk sac hemogenic endothelium over developmental time remains undefined. We performed a spatiotemporal analysis of EMP emergence, and document the morphological steps of the endothelial-to-hematopoietic transition. Emergence of rounded EMPs from polygonal clusters of Kit+ cells initiates prior to the establishment of arborized arterial and venous vasculature in the yolk sac. Interestingly, Kit+ polygonal clusters are detected in both arterial and venous vessels after remodeling. To determine whether there are similar mechanisms regulating the specification of EMPs with other angiogenic signals regulating adult-repopulating HSCs, we investigated the role of embryonic blood flow and Wnt/β-catenin signaling during EMP emergence. In embryos lacking a functional circulation, rounded Kit+ EMPs still fully emerge from unremodeled yolk sac vasculature. In contrast, canonical Wnt signaling appears to be a common mechanism regulating hematopoietic emergence from hemogenic endothelium. These data illustrate the heterogeneity in hematopoietic output and spatiotemporal regulation of primary embryonic hemogenic endothelium.