Pamela N. Pharr

Hemorrhage, impaired hematopoiesis, and lethality in mouse embryos carrying a targeted disruption of the Fli1 transcription factor.

ABSTRACT 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.

Defective Megakaryopoiesis and Abnormal Erythroid Development in Fli-1 Gene-Targeted Mice

Mouse embryos homozygous for a targeted disruption in theFli-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 ofFli-1 in the regulation of hematopoiesis and for c-mpl as aFli-1 target gene.

Erythropoietin Receptors That Signal Through Stat5 or Stat3 Support Fetal Liver and Adult Erythropoiesis: Lack of Specificity of Stat Signals During Red Blood Cell Development

Erythropoietin (Epo) is essential for formation of mature red blood cells (RBC). However, the function of Epo receptor (EpoR)-dependent signaling pathways in the regulation of erythropoiesis remains unclear. To determine whether specific Stat signals are required for RBC development, we changed the Stat signaling specificity of the EpoR. The wild-type EpoR activates only Stat5. Thus, we substituted the major Stat5 binding sites (residues 343 and 401) in the EpoR cytoplasmic region with the Stat3 binding/activation motif from gp130. We demonstrated that activated EpoRs containing a single substitution stimulate Stat5 and Stat3, whereas an EpoR with both substitutions stimulates Stat3 but not Stat5. We then determined the ability of these receptors to support fetal liver and adult erythropoiesis. Our results show that erythropoiesis is stimulated by EpoRs that activate Stat5, both Stat5 and Stat3, or Stat3 in place of Stat5. These findings demonstrate that the specificity of EpoR Stat signaling is not essential for RBC development.

Inference for an age-dependent multitype branching-process model of mast cells

We consider an age-dependent, multitype model for the growth of mast cells in culture. After a colony of cells is established by an initiator type, the two possible types of cells are resting and proliferative. Using novel inferential procedures, we estimate the generation-time distribution and the offspring distribution of proliferative cells, and the waiting-time distribution of resting cells.

The expression and role of human erythropoietin receptor in erythroid and nonerythroid cells.

During the past few years, our laboratory has investigated the erythropoietin receptor (EPOR) with regard to its structure-function relationships, role in development, transcriptional regulation, and the mechanism by which it conveys a signal upon interaction with its ligand. We began to focus on the EPOR as the result of three related developments. First, our recent studies indicated that erythropoietin (EPO) functioned as a viability factor rather than as an inducer of erythropoiesis.

Signaling by the Erythropoietin receptor in retrovirally transduced normal hematopoietic progenitors: A conserved proline-rich motif is required for association with JAK2

The receptor for erythropoietin is restricted to cells of mature erythroid and possibly megakaryocyte lineages. Studies in cell lines have suggested that cytokine receptors share a conserved signaling pathway for proliferation. We retrovirally transduced the erythropoietin receptor or a constitutively activated form of the EPOR into normal hematopoietic progenitors, including blast cell colonies. The EPO-R was able to support the proliferation and differentiation of early erythroid, early megakaryocytic, and macrophage progenitors, but not granulocyte progenitors. Blast cell colonies transduced with the EPO-R proliferate in response to EPO but the development of erythroid cells was not favored over other lineages. These results with normal cells suggest that some but not all cytokine receptors exhibit shared signaling pathways, and that EPO signaling alone is not sufficient to drive erythroid development. The Janus family of cytosolic tyrosine kinases mediate cytokine initiated mitogenic signals. We have determined that the EPO-R box 1 cytoplasmic motif is required for the binding and activation of JAK2. However, sequences outside the box 1 domain most likely regulate the specificity of JAK kinase association.