The receptor tyrosine kinase Flk-1 (ref. 1) is believed to play a pivotal role in endothelial development. Expression of the Flk-1 receptor is restricted to endothelial cells and their embryonic precursors, and is complementary to that of its ligand, vascular endothelial growth factor (VEGF), which is an endothelial-specific mitogen. Highest levels of flk-1 expression are observed during embryonic vasculogenesis and angiogenesis, and during pathological processes associated with neovascularization, such as tumour angiogenesis. Because flk-1 expression can be detected in presumptive mesodermal yolk-sac blood-island progenitors as early as 7.0 days postcoitum, Flk-1 may mark the putative common embryonic endothelial and haematopoietic precursor, the haemangioblast, and thus may also be involved in early haematopoiesis. Here we report the generation of mice deficient in Flk-1 by disruption of the gene using homologous recombination in embryonic stem (ES) cells. Embryos homozygous for this mutation die in utero between 8.5 and 9.5 days post-coitum, as a result of an early defect in the development of haematopoietic and endothelial cells. Yolk-sac blood islands were absent at 7.5 days, organized blood vessels could not be observed in the embryo or yolk sac at any stage, and haematopoietic progenitors were severely reduced. These results indicate that Flk-1 is essential for yolk-sac blood-island formation and vasculogenesis in the mouse embryo.

Cell surface antigen CD109 is a glycosylphosphatidylinositol (GPI)-linked glycoprotein of approximately 170 kd found on a subset of hematopoietic stem and progenitor cells and on activated platelets and T cells. Although it has been suggested that T-cell CD109 may play a role in antibodyinducing T-helper function and it is known that platelet CD109 carries the Gov alloantigen system, the role of CD109 in hematopoietic cells remains largely unknown. As a first step toward elucidating the function of CD109, we have isolated and characterized a human CD109 cDNA from KG1a and endothelial cells. The isolated cDNA comprises a 4335 bp open-reading frame encoding a 1445 amino acid (aa) protein of approximately 162 kd that contains a 21 aa Nterminal leader peptide, 17 potential Nlinked glycosylation sites, and a C-terminal GPI anchor cleavage-addition site. We report that CD109 is a novel member of the ␣2 macroglobulin (␣2M)/C3, C4, C5 family of thioester-containing proteins, and we demonstrate that native CD109 does indeed contain an intact thioester. Analysis of the CD109 aa sequence suggests that CD109 is likely activated by proteolytic cleavage and thereby becomes capable of thioester-mediated covalent binding to adjacent molecules or cells. In addition, the predicted chemical reactivity of the activated CD109 thioester is complementlike rather than resembling that of ␣2M proteins. Thus, not only is CD109 potentially capable of covalent binding to carbohydrate and protein targets, but the t ½ of its activated thioester is likely extremely short, indicating that CD109 action is highly restricted spatially to the site of its activation. IntroductionTo identify new surface antigens expressed by primitive hematopoietic stem and progenitor cells, we raised a series of monoclonal antibodies (mAbs) against the primitive CD34 ϩ acute myeloid leukemia cell line, KG1a. 1-3 Four of these mAbs-8A3, 7D1, 8A1, and 7C5-recognized a novel glycoprotein of approximately 170 kd that was expressed in a restricted pattern in the hematopoietic compartment and by endothelial cells. 4 Subsequently found to be identified by a number of additional mAbs, this antigen was designated CDw109 in 1993 5 and CD109 in 1996. 6 Antibodies to CD109 recognize monomeric polypeptides of about 170 kd and 150 kd in lysates of KG1a cells, T-cell lines, and activated T lymphoblasts, endothelial cells, and activated platelets. 3,[7][8][9][10][11] Peptide mapping and amino acid (aa) analysis indicate that the 150-kd form is likely derived proteolytically from the 170-kd form. 3,10 An additional band of about 120 kd that is occasionally observed arises through calcium-dependent proteolysis of the larger forms. 3,10 CD109 contains several N-linked endoglycosidase H-sensitive hybrid-type glycans but no O-linked glycans. 3,10 Consistent with this finding, ABH blood group antigens have recently been shown to be carried by platelet CD109. 12 KG1a CD109 is susceptible to cleavage with phosphatidylinositolspecific phospholipase C (PI-PLC), indicating that CD109 is bo...