The generation of blood cells, haematopoiesis, in the mouse embryo begins with the development of primitive nucleated erythroid cells in the yolk sac followed by the appearance of precursors for multiple definitive haematopoietic lineages. The later developing lineages arise from multipotential stem cells, but the relationship of primitive erythroid cells to these other haematopoietic populations is unknown. Using an in vitro embryonic stem (ES) cell differentiation system, we show that primitive erythrocytes and other haematopoietic lineages arise from a common multipotential precursor that develops within embryoid bodies generated from differentiated ES cells. In response to vascular endothelial growth factor and c-kit ligand these precursors give rise to colonies containing immature cells (blasts) expressing marker genes characteristic of haematopoietic precursors. Many blast colonies also expressed betaH1 and beta major globins but not Brachyury, a mesodermal marker. Kinetic analysis demonstrated that the blast colony-forming cells represent a transient population, preceding the establishment of the primitive erythroid and other lineage-restricted precursors. This precursor population may represent the earliest stage of embryonic haematopoietic commitment.

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We describe the construction of a v‐rel estrogen receptor fusion protein (v‐relER) which allows the regulation of v‐rel oncoprotein activity by hormone. In the presence of estrogen, v‐relER readily transformed primary chicken fibroblasts and bone marrow cells in vitro. In both cell types, v‐rel‐specific transformation was critically dependent on the presence of estrogen or the estrogen agonist 4‐hydroxytamoxifen (OHT). Withdrawal of estrogen or application of an estrogen antagonist, ICI164,384 (ICI) caused a reversal of the transformed phenotype. We also demonstrate that the v‐relER protein binds to NF‐kappa B sites in an estrogen‐dependent manner, thereby showing that sequence‐specific DNA binding of v‐relER is critical for the activation of its transforming capacity. In transient transfection experiments, we failed to demonstrate a clear repressor or activator function of the v‐rel moiety in v‐relER. However, in v‐relER‐transformed bone marrow cells, estrogen and OHT induced elevated mRNA levels of two cellular genes whose expression is constitutive and high in v‐rel‐transformed cells. These results suggest that v‐rel might exert part of its activity as an activator of rel‐responsive genes.

The avian reticuloendotheliosis virus T contains within its genome the oncogene rel. The expression of this gene is responsible for the induction of lymphoid tumors in birds. Recently, the rel gene was shown to be related to the p50 DNA binding subunit of the transcription factor complex NF-KB. Binding sites for the NF-#cB complex are found in the enhancer regions of a number of genes, including the immunoglobulin K gene and the human immunodeficiency virus long terminal repeat. In this communication we identify an activity from avian reticuloendotheliosis virus T-transformed avian lymphoid cells that binds in an electrophoretic-mobility-shift assay to an NF-KB binding site from the K enhancer. This activity contains proteins immunologically related to rel, as detected by polyclonal and monoclonal antibodies directed against v-rel. In a DNA affinity precipitation assay using the NF-KB site from the human immunodeficiency virus long terminal repeat, v-rel and several other proteins were identified. These data suggest that oncogenic transformation by v-rel is the result of an altered pattern of gene expression.Infection of birds with the avian reticuloendotheliosis virus T (REV-T) results in a fatal lymphoma within days after infection (1). In vitro REV-T is also able to transform avian lymphoid cells (2). v-rel, the oncogene harbored by REV-T, is a truncated form of its cellular homologue, c-rel, that lacks two amino acids at the amino terminus and 118 amino acids at the carboxyl terminus (3, 4). The protein product of the v-rel gene p59v-rel is a phosphoprotein that is found in both the nucleus and cytoplasm in transformed lymphoid cells (5). We (5) and others (6) have demonstrated that v-rel is part ofa high molecular mass complex in the infected cell that includes cellular proteins of 124 kDa, 115 kDa, and 36 kDa.The rel gene was shown to be related to the Drosophila embryonic polarity gene dorsal. The extent of homology in a 295-amino acid stretch at the amino terminus is 75% when conservative amino acid changes are taken into account (7). The carboxyl termini are unrelated. Reminiscent of v-rel, the dorsal protein is also found either in the cytoplasm or nucleus. However, the localization of dorsal is controlled by positional effects within the developing embryo and carboxyl-terminal sequences (8). The recent cloning of the p50 DNA binding subunits ofthe transcription factors NF-KB and KBF-1 also revealed homology with rel (9, 10). As with dorsal, the homology extends over -334 amino acids at the amino terminus. Biochemical evidence suggests further similarities between these proteins. NF-KB is found in the cytoplasm and nucleus and also associates in complex with other cellular proteins (11).Functional studies support the suggestion that NF-KB, dorsal, and rel are closely related. All three proteins are able to modulate transcription from a variety of promoters (11)(12)(13). In addition, the c-rel protein contains a carboxyl-terminal transcriptional activation domain as shown by fusion to LexA or GA...

Avian leukosis virus (ALV)-induced neoplasias are commonly found associated with integrations of proviral DNA in proximity to the myc gene. However, studies suggest that other genetic events are necessary for the complete neoplastic phenotype. A cell line (HP46) derived from an ALV-induced tumor has been analyzed and found to contain, in addition to an alteration in the myc gene, a promoter insertion in the c-rel locus. Both loci expressed large amounts of mRNA coding for their respective proteins. Several rel-related transcripts were expressed in the HP46 line, and four rel-related proteins of lower molecular weight than the wild-type p68cre1 product were detected. At least two of these transcripts contained U5 long terminal repeat sequences on the 5' end of the RNA. Structural data suggest that the messages may have evolved by an alternative splicing mechanism. This is the first example of a promoter insertion in the c-rel locus, a gene whose viral counterpart v-rel is responsible for the induction of lymphoid tumors.

We have investigated the expression pattern of the Flk-1 receptor tyrosine kinase in mouse embryonic and fetal hematopoietic tissues as well as on hematopoietic precursor cells derived from these tissues. RNA analysis indicated that flk-1 was expressed in the yolk sac at day 10 of gestation, in the whole embryo at day 10 and 12 of gestation, in the liver throughout fetal life and in embryoid bodies (EBs) generated from ES cells differentiated in culture. Flk-1 message was also detected in erythroid and macrophage colonies generated from precursors of yolk sac, fetal liver, adult marrow and EB origin. Using an antibody directed against the extracellular portion of the molecule we have found that up to 50% of cells from EBs differentiated for 4 days express Flk-1. Following the development of this early Flk-1+ population the number of receptor-positive cells declines progressively to represent less than 5% of the EBs by day 12 of differentiation. Kinetic analysis revealed that the establishment of the EB Flk-1+ population precedes the development of cells which express CD34, Ly6A (Sca-1) and AA4.1. Cell sorting experiments demonstrated that all day-4 EB-derived hematopoietic precursors are Flk-1+ whereas greater than 95% of those found within the day-12 EBs are Flk-1-, suggesting that the precursor population which expresses this receptor represents an early but transient wave of hematopoietic development. Analysis of yolk sac and whole embryos at day 8.5 of gestation revealed a small but distinct Flk-1+ population that contained hematopoietic precursors. Day-12.5 fetal liver contained few Flk-1+ cells that showed little hematopoietic potential. Together these findings indicate that Flk-1 is expressed on an early population of hematopoietic precursors that may represent the onset of embryonic hematopoiesis.

Avian leukosis virus (ALV)-induced neoplasias are commonly found associated with integrations of proviral DNA in proximity to the myc gene. However, studies suggest that other genetic events are necessary for the complete neoplastic phenotype. A cell line (HP46) derived from an ALV-induced tumor has been analyzed and found to contain, in addition to an alteration in the myc gene, a promoter insertion in the c-rel locus. Both loci expressed large amounts of mRNA coding for their respective proteins. Several rel-related transcripts were expressed in the HP46 line, and four rel-related proteins of lower molecular weight than the wild-type p68c-rel product were detected. At least two of these transcripts contained U5 long terminal repeat sequences on the 5' end of the RNA. Structural data suggest that the messages may have evolved by an alternative splicing mechanism. This is the first example of a promoter insertion in the c-rel locus, a gene whose viral counterpart v-rel is responsible for the induction of lymphoid tumors.