The Ets family of transcription factors have been suggested to function as key regulators of hematopoeisis. Here we describe aberrant hematopoeisis and hemorrhaging in mouse embryos homozygous for a targeted disruption in the Ets family member, Fli1. Mutant embryos are found to hemorrhage from the dorsal aorta to the lumen of the neural tube and ventricles of the brain (hematorrhachis) on embryonic day 11.0 (E11.0) and are dead by E12.5. Histological examinations and in situ hybridization reveal disorganization of columnar epithelium and the presence of hematomas within the neuroepithelium and disruption of the basement membrane lying between this and mesenchymal tissues, both of which express Fli1 at the time of hemorrhaging. Livers from mutant embryos contain few pronormoblasts and basophilic normoblasts and have drastically reduced numbers of colony forming cells. These defects occur with complete penetrance of phenotype regardless of the genetic background (inbred B6, hybrid 129/B6, or outbred CD1) or the targeted embryonic stem cell line used for the generation of knockout lines. Taken together, these results provide in vivo evidence for the role of Fli1 in the regulation of hematopoiesis and hemostasis.The human FLI1 gene, which we originally cloned from the leukemia T-cell line, CEM, is a member of the Ets gene family of transcription factors (38). As observed with all other members of the Ets gene family, FLI1 encodes a protein that retains a region of conserved sequence, the Ets domain (37, 38). This minimal 85-amino-acid region has been shown to be the DNAbinding domain. Ets proteins bind to DNA sequences that contain a consensus GGA(A/T) core motif (Ets-binding site) and, in the majority of cases, function as transcriptional activators. Ets proteins control the expression of genes that are critical for the control of cellular proliferation, differentiation, and programmed cell death. The presence of multiple Ets family proteins in a variety of cell types and the overlapping DNA-binding specificity of the Ets proteins have made it difficult to identify target genes that are specific for individual Ets factors. The generation and analysis of targeted disruptions in individual family members, coupled with the identification of such target genes, however, is one approach to understanding the role of Ets transcription factors in normal and dysregulated development. A variety of studies including the analysis of expression of members of the Ets transcription factor family in hematopoietic tissues and cell lines and the generation and analysis of targeted mutations in Ets gene family members in mouse suggest that they play important roles in the regulation of normal hematopoietic development (13,14,29).FLI1 was found to be highly related to the human ERG gene, and we originally named it ERGB to reflect this homology (38). Sequence alignments of the predicted 452-amino-acid protein product of human FLI1 with those of the ERG and mouse Fli1 products (5) demonstrated 80 and 96% similarity, respectively. FLI1 ha...

Mouse embryos homozygous for a targeted disruption in the Fli-1 gene show hemorrhage into the neural tube and brain on embryonic day (E)11.0 and die shortly thereafter. Livers from the mutant embryos contain drastically reduced numbers of pronormoblasts, basophilic normoblasts, and colony-forming cells. To determine the nature of impaired hematopoiesis, we carried out cell culture studies of mutant embryonic stem (ES) cells and cells from the aorta-gonad-mesonephros (AGM) region of E10.0 mutant embryos. There was a striking reduction in the number of megakaryocytes in cultures of mutant AGM cells compared with cultures of AGM cells from wild-type or heterozygous embryos. Furthermore, Fli-1 mutant ES cells failed to produce megakaryocyte colonies and multilineage colonies containing megakaryocytes. Consistent with the observed defect in megakaryopoiesis, we also demonstrated the down-regulation of c-mpl in the AGM of mutant embryos. The percentages of pronormoblasts and basophilic normoblasts were significantly reduced in cultures of mutant AGM embryos, which contained primarily polychromatophilic and orthochromatic normoblasts. These results provide further evidence for the role of Fli-1 in the regulation of hematopoiesis and for c-mpl as a Fli-1 target gene.

As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated interleukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. We now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content :50-fold and their carboxypeptidase A content =lOO-fold, and augment =45-fold their biosynthesis of proteoglycans bearing 35S-labeled heparin relative to 35S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment.Cloned and noncloned mouse mast cells have been obtained by culturing progenitors from bone marrow, spleen, lymph node, or fetal liver in conditioned medium that contains interleukin 3 (IL-3) (1-6). The IL-3-dependent mast cells obtained in vitro from these different tissue sources are characterized by failure to stain with safranin, preferential synthesis of 35S-labeled chondroitin sulfate E proteoglycans (7,8), low content of histamine (1-6), and low content of carboxypeptidase A (see below).The connective tissue mast cell, represented by the mouse or rat serosal mast cell, is safranin+, preferentially synthesizes 35S-labeled heparin proteoglycans (7, 9), has high levels of granular histamine (10) and carboxypeptidase A (11), and can be maintained ex vivo by coculture with mouse 3T3 fibroblasts (12). Recently we demonstrated that noncloned, IL-3-dependent bone marrow-derived mast cells cocultured with fibroblasts for 7-14 days changed their histochemical properties from safranin-to safranin+, increased their histamine content =15-fold, and augmented their rate of synthesis of 35S-labeled heparin proteoglycans (13). Because these mast cells were not cloned, whether fibroblasts selectively promoted expansion of a minor subpopulation of safranin+ mast cells or altered the phenotype of the predominant population of cells could not be decided. By using cloned mast cells, we establish in the present study that coculture with fibroblasts induces changes in the entire population of safranin-mast cells. Phenotypic changes were seen in all three major classes of constituents of the secretory granule-namely, histamine, proteoglycan, and neutral protease. MATERIALS AND METHODSCulture of Mast Cells with and without Fibroblasts. Spleen cells (2 x 105) from 10-to 15-wk-old female BDF1 mice (Simonsen Laboratories, Gilroy, CA) were suspended in 1 ml of alpha-modified medium (Flow Laboratories, Rockville, MD) containing 30% fetal calf serum (Flow Laboratories), 1% deionized bovine serum albumin (Calb...