INTRODUCTION
The first hematopoietic and endothelial precursors arise from extra-embryonic mesoderm and differentiate to form the blood islands in the yolk sac of the early embryo (Moore and Metcalf, 1970
). The close spatial and temporal development of these lineages within the blood islands provided the basis for the hypothesis that they arise from a common progenitor, a cell known as the hemangioblast (Sabin, 1920; Wagner, 1980). Indirect evidence in support of the concept of the hemangioblast has come from studies demonstrating that the hematopoietic and endothelial lineages express a large number of different genes in common and that some of these genes are essential for both blood cell and vascular development (Anagnostou et al., 1994; Asahara et al., 1997; Fina et al., 1990; Kabrun et al., 1997; Kallianpur et al., 1994; Millauer et al., 1993; Porcher et al., 1996; Robb et al., 1995; Shalaby et al., 1995; Yamaguchi et al., 1993; Young et al., 1995).
Direct demonstration for the existence of a progenitor with hemangioblast properties has been provided by experiments using a model system based on the in vitro differentiation potential of embryonic stem (ES) cells (Choi et al., 1998; Nishikawa et al., 1998). Following the initiation of differentiation in culture, ES cells will form colonies known as embryoid bodies (EBs), that generate hematopoietic and endothelial progeny in a temporal pattern recapitulating the development of these populations in the yolk sac (Keller et al., 1993; Palis et al., 1999; Vittet et al., 1996). Analysis of early EBs, prior to the hematopoietic and endothelial commitment stages, revealed the presence of a progenitor with hemangioblast potential. In response to VEGF, these progenitors generate blast colonies that display both hematopoietic and endothelial potential (Choi et al., 1998). Kinetic studies demonstrated that this progenitor or blast colony-forming cell (BL-CFC) represents a transient population that is present within the EBs for approximately 36 hours, between day 2.5 and 4 of differentiation, preceding the onset of primitive erythropoiesis. The characteristics of the BLCFC, namely its early development and its potential to generate primitive and/or definitive hematopoietic as well as endothelial progeny, suggests that it represents the in vitro equivalent of the yolk sac hemangioblast. More recent studies have shown that most BL-CFC express Flk1 (VEGF receptor 2; Kdr – Mouse Genome Informatics)) and that a subpopulation of Flk1+ cells also expresses the transcription factor Scl (Chung et al., 2002; Faloon et al., 2000). The early development of the BL-CFC, prior to hematopoietic commitment, suggests that it could be a direct descendent of a mesodermal progenitor, or possibly a subpopulation of mesoderm. In the ES/EB model system, mesoderm, as defined by expression of the T-box gene brachyury, is induced within 48 hours of the onset of differentiation and persists until day 4 (Robertson et al., 2000). This mesodermal window overlaps with the onset of Flk1 expression, initiated as early as day 2.5 of differentiation and with the BL-CFC stage of development, typically found between day 2.5 and 4.0 of differentiation.
In the mouse embryo, mesoderm is generated from the epiblast or embryonic ectoderm through the process of gastrulation that is initiated at approximately day 6.5 of gestation (reviewed by Tam and Behringer, 1997). At the onset of gastrulation, the epiblast cells in the region that defines the posterior part of the embryo undergo an epithelial to mesenchymal transition and form a transient structure known as the primitive streak from which the mesoderm emerges. The newly formed mesoderm migrates away from the primitive streak, moves laterally and anteriorly and is patterned into various populations with distinct developmental fates. Brachyury is expressed in all nascent mesoderm and downregulated as these cells undergo patterning and specification into the derivative tissues including skeletal muscle, cardiac muscle and connective tissues in addition to blood and endothelium (Herrmann, 1991; Kispert and Herrmann, 1994).
The first mesodermal cells to develop within the embryo contribute predominantly to the extra-embryonic tissues, giving rise to the hematopoietic and vascular cells of the yolk sac (Kinder et al., 1999). Hematopoietic progenitors are first found in the developing yolk sac as early as day 7.0 of gestation, 
12 hours after the beginning of gastrulation (Palis et al., 1999). Flk1 is expressed in the yolk sac at this stage and is essential for the establishment of the blood cell and vascular lineages (Schuh et al., 1999; Shalaby et al., 1995). Although the yolk sac hemangioblast has not yet been identified, the rapid commitment to the hematopoietic and endothelial lineages following the induction of mesoderm suggests that this putative in vivo progenitor should also be closely related to mesoderm.
Our understanding of hematopoietic and endothelial development has been greatly enhanced by the identification and characterization of cell populations representing the earliest stages of commitment towards these lineages (Nishikawa et al., 1998; Chung et al., 2002; Faloon et al., 2000; Lacaud et al., 2002; Robertson et al., 2000). By contrast, however, developmental stages earlier than the hemangioblast remain difficult to study as there are few known cell surface markers that enable one to isolate these populations or subsets of progenitors within these populations. To access prehemangioblast cell populations and define their relationship with respect to the BL-CFC, we targeted the GFP cDNA to the brachyury locus. In this report, we show that GFP is an effective marker for the mesodermal populations that develop within the EBs. Analysis of GFP and Flk1 expression led to the identification of three subpopulations GFP–Flk1–, GFP+Flk1– and GFP+Flk1+ that represent a developmental progression from pre-mesodermal cells to the hemangioblast.
Answers to all your Biology Questions
