Hematopoiesis in the early embryo initiates at two independent sites, the yolk sac and an intraembryonic region known as the para-aortic splanchnopleura (P-Sp), which later contains the developing aorta, gonads, and mesonephros (AGM) (Dieterlen-Lievre 1975
; Russel 1979; Godin et al. 1995; Medvinsky and Dzierzak 1996; Palis et al. 1999). Detailed analysis of hematopoietic development in the early embryo strongly suggests that the programs generated in these two regions are different. Hematopoietic commitment is detected first in the yolk sac, where distinct blood islands appear, shortly following gastrulation (Moore and Metcalf 1970; Haar and Ackerman 1971; Palis et al. 1995, 1999). These blood islands consist of an inner cluster of maturing erythrocytes surrounded by a layer of developing endothelial cells (Haar and Ackerman 1971). The erythroid cells within these blood islands, known as primitive erythrocytes, are distinct from fetal and adult erythrocytes in that they are large, circulate in the bloodstream as nucleated cells for much of their life span, and contain an embryonic form of hemoglobin (Barker 1968; Brotherton et al. 1979; Russel 1979; Kingsley et al. 2004). Production of primitive erythrocytes is known as primitive erythropoiesis and is restricted to the yolk sac during a narrow window of development in the mouse embryo (Palis et al. 1999). Development of all other blood cell lineages including myeloid, fetal, and adult erythroid and lymphoid is referred to as definitive hematopoiesis. Definitive erythroid cells enucleate prior to entering the bloodstream, are smaller than those of the primitive lineage, and produce adult forms of hemoglobin.
In addition to primitive erythrocytes, the yolk sac generates a subset of lineages from the definitive hematopoietic program including the macrophage, definitive erythroid, and mast cell (Palis et al. 1999). Kinetic analysis of the developing yolk sac revealed that these lineages are produced in a defined temporal pattern with primitive erythroid and macrophage appearing first, followed by definitive erythroid, which, in turn, is followed by mast cells. While the yolk sac does have potential beyond that of primitive erythropoiesis, it does not appear to be capable of generating lymphocytes or HSCs, when analyzed prior to the onset of circulation (Cumano et al. 2001). Parallel studies have demonstrated that the hematopoietic program initiated in the P-Sp includes the generation of the myeloid, lymphoid, and definitive erythroid lineages as well as the HSCs (Muller et al. 1994; Godin et al. 1995; Cumano et al. 2001). The P-Sp does not, however, generate primitive erythrocytes. Thus, the distinguishing features of the early yolk sac are the generation of the primitive erythroid lineage and a lack of lymphoid and HSC potential, while the P-Sp program can be defined by the development of the lymphoid lineages and HSCs. Collectively, these observations indicate that the hematopoietic system initiates with the production of a limited number of specialized lineages in the yolk sac and matures with time into a full multilineage system with the switch to the P-Sp. While somewhat unusual, this pattern is logical, as the system is responding to the requirements of the embryo at different developmental stages. These dramatic changes in the hematopoietic system, in particular the early and transient appearance of the primitive erythroid lineage, provide a developmental map for monitoring hematopoietic commitment in the ES cell differentiation cultures.
ES-cell-derived primitive and definitive hematopoiesis
In optimized culture conditions following serum induction, ES cells will undergo hematopoietic differentiation (Keller 1995). Hematopoietic commitment within these cultures can be easily monitored by gene expression patterns (Schmitt et al. 1991; Keller et al. 1993; Robertson et al. 2000), the appearance of specific cell surface markers (Kabrun et al. 1997; Nishikawa et al. 1998), and the development of clonable progenitor cells (Schmitt et al. 1991; Keller et al. 1993). With these assays, it has been possible to demonstrate that development of the hematopoietic lineages is highly reproducible and efficient. Under appropriate culture conditions, >50% of the cells in the differentiation cultures will express the hematopoietic/vascular receptor tyrosine kinase Flk-1 (VEGF receptor 2) (Kabrun et al. 1997) and up to 5% can represent a clonable hematopoietic progenitor (Keller et al. 1993). Detailed analyses of the early stages of hematopoietic commitment have shown that both gene expression patterns and the kinetics of lineage development within EBs accurately reflect that found in the yolk sac (Keller et al. 1993; Palis et al. 1999; Robertson et al. 2000). Most notable was the finding that the primitive erythroid lineage develops earliest and represents a transient population that persists in the EBs for 
4 d (Keller et al. 1993). The macrophage, definitive erythroid, and mast cell lineages appear following the onset of primitive erythropoiesis and develop in the temporal order found in the yolk sac (Keller et al. 1993). Lymphoid progenitors and HSCs have not been identified among the progeny of early stage EBs, suggesting that the initial stages of EB hematopoiesis represent the equivalent of yolk sac hematopoiesis.
The faithful recapitulation of this yolk sac developmental program provides strong evidence that regulation of hematopoietic commitment in the ES/EB model is similar, if not identical, to that of the early embryo. Support for this interpretation has been provided by gene targeting studies that helped define the role of specific transcription factors including Scl/tal-1 (Begley et al. 1989), Runx1 (Wang and Speck 1992; Ogawa et al. 1993), and GATA-1 (Orkin 1992) in the establishment of the hematopoietic system. Each of these factors functions at specific stages of blood cell differentiation as demonstrated by the observations that Scl/tal-1 is required for the development of all hematopoietic (primitive erythroid and definitive) lineages (Robb et al. 1995; Shivdasani et al. 1995), Runx1 for the definitive lineages but not primitive erythropoiesis (Okuda et al. 1996; Wang et al. 1996), and GATA-1 for late-stage primitive and definitive erythroid maturation (Pevny et al. 1991; Weiss et al. 1994). All of these defects have been accurately replicated in the ES cell differentiation model (Weiss et al. 1994; Porcher et al. 1996; Wang et al. 1996; Lacaud et al. 2002). In addition to further validating the ES cell system as a model of hematopoietic development, the ability to analyze mutations in EBs provides a powerful model for structure/function studies as well as for the identification of molecular targets of the gene of interest.
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