Erythroid Krüppel-like factor (EKLF) was originally isolated from erythroid cell RNA by differential screening and shown to be erythroid-specific, although a low level of EKLF was found in mast cell lines. EKLF contains three zinc-fingers homologous to those found in the Krüppel family of transcription factors. Because it binds the sequence CCACACCCT, EKLF may affect erythroid development as a result of its ability to bind to the CAC box in the promoter of the beta-globin gene. Mutation of this element leads to reduced beta-globin expression and it appears to mediate the effect of the globin locus control region on the promoter. Here we inactivate the EKLF gene through insertion of a lacZ reporter gene by homologous recombination in embryonic stem (ES) cells. Heterozygous EKLF+/- mice show that the reporter gene is expressed in a developmentally specific manner in all types of erythroblasts in the fetal liver and adult bone marrow. Homozygous EKLF-/- mice appear normal during the embryonic stage of haematopoiesis in the yolk sac, but develop a fatal anaemia during early fetal life when haematopoiesis has switched to the fetal liver. Enucleated erythrocytes are formed but these do not contain the proper amount of haemoglobin. We conclude that the transcription factor EKLF is essential for the final steps of definitive erythropoiesis in fetal liver.

GATA-2 is an essential transcription factor in the hematopoietic system that is expressed in hematopoietic stem cells (HSCs) and progenitors. Complete deficiency of GATA-2 in the mouse leads to severe anemia and embryonic lethality. The role of GATA-2 and dosage effects of this transcription factor in HSC development within the embryo and adult are largely unexplored. Here we examined the effects of GATA-2 gene dosage on the generation and expansion of HSCs in several hematopoietic sites throughout mouse development. We show that a haploid dose of GATA-2 severely reduces production and expansion of HSCs specifically in the aorta-gonad-mesonephros region (which autonomously generates the first HSCs), whereas quantitative reduction of HSCs is minimal or unchanged in yolk sac, fetal liver, and adult bone marrow. However, HSCs in all these ontogenically distinct anatomical sites are qualitatively defective in serial or competitive transplantation assays. Also, cytotoxic drug-induced regeneration studies show a clear GATA-2 dose–related proliferation defect in adult bone marrow. Thus, GATA-2 plays at least two functionally distinct roles during ontogeny of HSCs: the production and expansion of HSCs in the aorta-gonad-mesonephros and the proliferation of HSCs in the adult bone marrow.

Recently it was shown that, analogous to normal hematopoietic cells, the level of CXC chemokine receptor 4 (CXCR-4) expression on acute myeloid leukemia (AML) cells correlates with stromal cell derived factor-1 alpha (SDF-1)-induced chemotaxis. As we speculated that an anomalous organ distribution of AML cells could affect cell survival and thus result in an altered fraction surviving chemotherapy, we examined a possible correlation between patient prognosis and CXCR-4 expression in AML patients. We found that patients with a high CXCR-4 expression in the CD34 ؉ subset had a significantly reduced survival and a higher probability of relapse, resulting in a median relapsefree survival (

Internal tandem duplications of the Flt3 gene (Flt3/ITDs) are present in about 18% of all AML cases and are therefore one of the most frequent somatic gene mutations in AML. Little is known about the role of Flt3/ITDs in leukemogenesis or their clinical relevance. In this study we compared 18 samples with Flt3/ITDs and 63 AML samples without these mutations with respect to clinical prognosis, cytokine responsiveness, progenitor cell content and repopulation in the NOD/SCID mouse. We found that in patients with a mutation CR rates are reduced (P = 0.03) and relapse rates are increased (P = 0.01), indicating the prognostic importance of Flt3/ITDs. This is also emphasized by the finding that in patients under the age of 60 years, as well as in older patients the event-free survival was more unfavorable for the mutant patients (P = 0.003 and P = 0.03, respectively). At diagnosis Flt3/ITD and non-mutant AML bone marrow samples did not differ in their progenitor/stem cell frequencies. Cobblestone area forming cell (CAFC) subsets showed a similar frequency distribution in mutant and nonmutant samples. In 7-day liquid cultures, Flt3/ITD samples showed a reduced growth in response to a variety of myeloid growth factors. In contrast, Flt3/ITD samples displayed a higher ability to engraft the NOD/SCID bone marrow with leukemic cells. Together these data show that the Flt3/ITD represents an important diagnostic marker for patient prognosis, and that the presence of these mutations is associated with altered proliferative ability of progenitors in vivo and in vitro. Leukemia (2000) 14, 675-683.

The aorta-gonads-mesonephros (AGM) region autonomously generates the first adult repopulating hematopoietic stem cells (HSCs) in the mouse embryo. HSC activity is initially localized to the dorsal aorta and mesenchyme (AM) and vitelline and umbilical arteries. Thereafter, HSC activity is found in the urogenital ridges (UGs), yolk sac, and liver. As increasing numbers of HSCs are generated, it is thought that these sites provide supportive microenvironments in which HSCs are harbored until the bone marrow microenvironment is established. However, little is known about the supportive cells within these midgestational sites, and particularly which microenvironment is most supportive for HSC growth and maintenance. Thus, to better understand the cells and molecules involved in hematopoietic support in the midgestation embryo, more than 100 stromal cell lines and clones were established from these sites. Numerous stromal clones were found to maintain hematopoietic progenitors and HSCs to a similar degree as, or better than, previously described murine stromal lines. Both the AM and UG subregions of the AGM produced many supportive clones, with the most highly HSCsupportive clone being derived from the UGs. Interestingly, the liver at this stage yielded only few supportive stromal clones. These results strongly suggest that during midgestation, not only the AM but also the UG subregion provides a potent microenvironment for growth and maintenance of the first HSCs. IntroductionThroughout adult life, the hematopoietic hierarchy is derived from hematopoietic stem cells (HSCs) maintained in the supportive microenvironment of the bone marrow (BM). Functional blood cells arise through a series of differentiation steps first occurring within the HSCs and proceeding through a hierarchy of progenitor cell types with increasing lineage commitment. 1-3 Both maintenance and differentiation of HSCs are controlled by complex interactions with the stromal microenvironment, 4 which consists of several morphologically distinct cell types including myofibroblasts and macrophages.Efforts to examine the interactions between HSCs and the microenvironment have led to the establishment of in vitro culture systems with adherent cells from BM. 5,6 Further studies have demonstrated that BM and fetal liver stromal cells maintain hematopoietic progenitors, long-term culture-initiating cells, cobblestone area-forming cells, and HSCs (cells capable of permanently repopulating the entire hematopoietic system of irradiated adult recipients). 7-9 Numerous stromal cell lines of adult BM and fetal liver hematopoietic microenvironments have been cloned and characterized for the growth, maintenance, and differentiation of HSCs 8-10 (see also references in Remy-Martin et al 11 ). Generally, these studies show an initial decrease in HSC activity 12 followed by an expansion phase of immature hematopoietic progenitors. 13 Most notably, the AFT024 stromal clone, derived from mouse fetal liver at day 14.5 of gestation, shows the best continued maintenance of...