SCL/tal-1-dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation

Hematopoiesis in most vertebrate species occurs in two distinct phases, primitive and definitive, which diverge from FLK1(+)VE-cadherin(-) mesoderm and FLK1(+)VE-cadherin(+) endothelial cells (EC), respectively. This study aimed at determining the stage at which hematopoietic lineage fate is determined by manipulating the SCL/tal-1 expression that is known to be essential for the early development of the primitive and definitive hematopoietic systems. We established SCL-null ES cell lines in which SCL expression is rescued by tamoxifen-inducible Cre recombinase-loxP site-mediated recombination. While no hematopoietic cells (HPC) were detected in SCL-null ES cell differentiation cultures, SCL gene reactivation from day 2 to day 4 after initiation of differentiation could rescue both primitive and definitive hematopoiesis. SCL reactivation at later phases was ineffective. Moreover, generation of VE-cadherin(+) EC that can give rise to definitive HPC required SCL reactivation prior to VE-cadherin expression. These results indicated that the competence to become HPC is acquired at the mesodermal stage by a SCL-dependent process that takes place independently of determination of endothelial fate.     

Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.

In this study, we have mapped the onset of hematopoietic development in the mouse embryo using colony-forming progenitor assays and PCR-based gene expression analysis. With this approach, we demonstrate that commitment of embryonic cells to hematopoietic fates begins in proximal regions of the egg cylinder at the mid-primitive streak stage (E7.0) with the simultaneous appearance of primitive erythroid and macrophage progenitors. Development of these progenitors was associated with the expression of SCL/tal-1 and GATA-1, genes known to be involved in the development and maturation of the hematopoietic system. Kinetic analysis revealed the transient nature of the primitive erythroid lineage, as progenitors increased in number in the developing yolk sac until early somite-pair stages of development (E8.25) and then declined sharply to undetectable levels by 20 somite pairs (E9.0). Primitive erythroid progenitors were not detected in any other tissue at any stage of embryonic development. The early wave of primitive erythropoiesis was followed by the appearance of definitive erythroid progenitors (BFU-E) that were first detectable at 1-7 somite pairs (E8.25) exclusively within the yolk sac. The appearance of BFU-E was followed by the development of later stage definitive erythroid (CFU-E), mast cell and bipotential granulocyte/macrophage progenitors in the yolk sac. C-myb, a gene essential for definitive hematopoiesis, was expressed at low levels in the yolk sac just prior to and during the early development of these definitive erythroid progenitors. All hematopoietic activity was localized to the yolk sac until circulation was established (E8.5) at which time progenitors from all lineages were detected in the bloodstream and subsequently in the fetal liver following its development. This pattern of development suggests that definitive hematopoietic progenitors arise in the yolk sac, migrate through the bloodstream and seed the fetal liver to rapidly initiate the first phase of intraembryonic hematopoiesis. Together, these findings demonstrate that commitment to hematopoietic fates begins in early gastrulation, that the yolk sac is the only site of primitive erythropoiesis and that the yolk sac serves as the first source of definitive hematopoietic progenitors during embryonic development.     

A GATA box in the GATA-1 gene hematopoietic enhancer is a critical element in the network of GATA factors and sites that regulate this gene

A region located at kbp -3.9 to -2.6 5' to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection analyses, this region activated reporter gene expression from an artificial promoter in a position- and orientation-independent manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent and also required the presence of the endogenous GATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1 gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL(-/-) embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls the expression of this gene.     

CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo

The platelet glycoprotein IIb (alpha(IIb); CD41) constitutes the alpha subunit of a highly expressed platelet surface integrin protein. We demonstrate that CD41 serves as the earliest marker of primitive erythroid progenitor cells in the embryonic day 7 (E7.0) yolk sac and high-level expression identifies essentially all E8.25 yolk sac definitive hematopoietic progenitors. Some definitive hematopoietic progenitor cells in the fetal liver and bone marrow also express CD41. Hematopoietic stem cell competitive repopulating ability is present in CD41(dim) and CD41(lo/-) cells isolated from bone marrow and fetal liver cells, however, activity is enriched in the CD41(lo/-) cells. CD41(bright) yolk sac definitive progenitor cells co-express CD61 and bind fibrinogen, demonstrating receptor function. Thus, CD41 expression marks the onset of primitive and definitive hematopoiesis in the murine embryo and persists as a marker of some stem and progenitor cell populations in the fetal liver and adult marrow, suggesting novel roles for this integrin.