PU.1 is a member of the ets family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. Mice homozygous for a disruption in the PU.1 DNA binding domain are born alive but die of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells and T cells, although erythrocytes and megakaryocytes were present. The absence of lymphoid commitment and development in null mice was not absolute, since mice maintained on antibiotics began to develop normal appearing T cells 3–5 days after birth. In contrast, mature B cells remained undetectable in these older mice. Within the myeloid lineage, despite a lack of macrophages in the older antibiotic‐treated animals, a few cells with the characteristics of neutrophils began to appear by day 3. While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.

No abstract

Members of the Ets family of transcription factors mediate transcriptional responses of multiple signaling pathways in diverse cell types and organisms. Targeted deletion of the conserved DNA binding domain of the Ets2 transcription factor results in the retardation and death of homozygous mouse embryos before 8.5 days of embryonic development. Defects in extraembryonic tissue gene expression and function include deficient expression of matrix metalloproteinase-9 (MMP-9, gelatinase B), persistent extracellular matrix, and failure of ectoplacental cone proliferation. Mutant embryos were rescued by aggregation with tetraploid mouse embryos, which complement the developmental defects by providing functional extraembryonic tissues. Rescued Ets2-deficient mice are viable and fertile but have wavy hair, curly whiskers, and abnormal hair follicle shape and arrangement, resembling mice with mutations of the EGF receptor or its ligands. However, these mice are not deficient in the production of TGF␣ or the EGF receptor. Homozygous mutant cell lines respond mitogenically to TGF␣, EGF, FGF1, and FGF2. However, FGF fails to induce MMP-13 (collagenase-3) and MMP-3 (stromelysin-1) in the Ets2-deficient fibroblasts. Ectopic expression of Ets2 in the deficient fibroblasts restores expression of both matrix metalloproteinases. Therefore, Ets2 is essential for placental function, mediating growth factor signaling to key target genes including MMP-3, MMP-9, and MMP-13 in different cell types, and for regulating hair development.

During mouse embryogenesis, macrophage-like cells arise first in the yolk sac and are produced subsequently in the liver. The onset of liver hematopoiesis is associated with the transition from primitive to definitive erythrocyte production. This report addresses the hypothesis that a similar transition in phenotype occurs in myelopoiesis. We have used whole mount in situ hybridization to detect macrophage-specific genes expressed during mouse development. The mouse c-fms mRNA, encoding the receptor for macrophage colony-stimulating factor (CSF-1), was expressed on phagocytic cells in the yolk sac and throughout the embryo before the onset of liver hematopoiesis. Similar cells were detected using the mannose receptor, the complement receptor (CR3), or the Microphthalmia transcription factor (MITF) as mRNA markers. By contrast, other markers including the F4/80 antigen, the macrophage scavenger receptor, the S-100 proteins, S100A8 and S100A9, and the secretory product lysozyme appeared later in development and appeared restricted to only a subset of c-fms–positive cells. Two-color immunolabeling on disaggregated cells confirmed that CR3 and c-fmsproteins are expressed on the same cells. Among the genes appearing later in development was the macrophage-restricted transcription factor, PU.1, which has been shown to be required for normal adult myelopoiesis. Mice with null mutations in PU.1 had normal numbers of c-fms–positive phagocytes at 11.5dpc. PU.1(−/−) embryonic stem cells were able to give rise to macrophage-like cells after cultivation in vitro. The results support previous evidence that yolk sac–derived fetal phagocytes are functionally distinct from those arising in the liver and develop via a different pathway.

The requirement of the transcription factor PU.1 for macrophage development has been well documented. However, the target genes regulated by PU.1 controlling macrophage maturation are not known. A granulocyte macrophage colony stimulating factor (GM-CSF)-dependent PU.1 null monocytic precursor cell was stably transduced with a PU.1-expressing retrovirus. The expression of PU.1 altered the surface expression of a few proteins expressed on monocytes; these cells, however, remained GM-CSF dependent and maintained an immature phenotype. In contrast to the PU.1 null cells, the cells expressing PU.1 responded to macrophage colony stimulating factor (M-CSF) with subsequent development into mature macrophages. Using suppressive subtractive hybridization between the PU.1 null and immature PU.1 rescued cells, three genes, MRP-14, Dap12 and CD53, were found expressed in the rescued cells, but not in the PU.1 null cells. In addition, these genes were modulated during M-CSF-induced maturation of the PU.1 rescued cells. The PU.1 null and rescued early monocytic cells provide a useful model to study the role of PU.1 in macrophage development.

PU.1 is a transcription factor shown to regulate the expression of many important genes in myeloid and B cells. At birth, mice homozygous for the disruption of the PU.1 gene have erythrocytes, megakaryocytes, and T cells, but no mature myeloid or B cells. Cells with an inability to develop to maturity were found in this mouse for B cells, neutrophils, eosinophils, mast cells, and monocytes. Rescue of early monocytic cells by transfection with the PU.1 gene results in renewed development to macrophages. These results demonstrate that PU.1 is an important regulator in the development of cells in the hematopoietic system.

Transcription factors play an important role choreographing lineage commitment and expansion of blood cells. Nuclear factors that are expressed primarily or exclusively in hematopoietic cells are likely candidates for regulating blood cell development. The transcription factor PU.1 is found only in hematopoietic cells, whereas ets-2, a related family member, is ubiquitously expressed. To compare the role of these two transcription factors in macrophage development, embryonic stem (ES) cells with a homozygous disruption of either the PU.1 or the ets-2 gene were generated. The ability of both knockout ES cells to differentiate to macrophages was tested. Normal development of macrophages, as determined by histochemical and immunohistochemical analysis, from PU.1 knockout ES cells was significantly blocked. Furthermore, the expression of known markers associated with macrophages, such as c-fms, CD11b, CD18 and granulocyte-macrophage colony-stimulating factor receptor, were not detected by reverse transcriptase-polymerase chain reaction. In contrast to the PU.1 knockout ES cells, macrophages, development from the ets-2 knockout ES cells was normal. Although both PU.1 and ets-2 are found in macrophages, these data show a distinct role for the lineage-restricted PU.1 transcription factor in macrophage development.

Mice homozygous for the disruption of the PU.1 (Spi-1) gene do not produce mature macrophages. In determining the role of PU.1 in macrophage differentiation, the present study investigated whether or not there was commitment to the monocytic lineage in the absence of PU.1. Early PU.1−/− myeloid colonies were generated from neonate liver under conditions that promote primarily macrophage and granulocyte/macrophage colonies. These PU.1−/− colonies were found to contain cells with monocytic characteristics as determined by nonspecific esterase stain and the use of monoclonal antibodies that recognize early monocyte precursors, including Moma-2, ER-MP12, ER-MP20, and ER-MP58. In addition, early myeloid cells could be grown from PU.1−/− fetal liver cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Similar to the PU.1 null colonies, the GM-CSF–dependent cells also possessed early monocytic characteristics, including the ability to phagocytize latex beads. The ability of PU.1−/− progenitors to commit to the monocytic lineage was also verified in vivo by flow cytometry and cytochemical analysis of primary neonate liver cells. The combined data shows that PU.1 is absolutely required for macrophage development after commitment to this lineage.