Bone marrow stem cells give rise to a variety of hematopoietic lineages and repopulate the blood throughout adult life. We show that, in a strain of mice incapable of developing cells of the myeloid ...and lymphoid lineages, transplanted adult bone marrow cells migrated into the brain and differentiated into cells that expressed neuron-specific antigens. These findings raise the possibility that bone marrow-derived cells may provide an alternative source of neurons in patients with neurodegenerative diseases or central nervous system injury.
Osteoclasts are multinucleated cells and the principal resorptive cells of bone. Although osteoclasts are of myeloid origin, the role of haematopoietic transcription factors in osteoclastogenesis has ...not been explored. Here we show that messenger RNA for the myeloid- and B-cell-specific transcription factor PU.1 progressively increases as marrow macrophages assume the osteoclast phenotype in vitro. The association between PU.1 and osteoclast differentiation was confirmed by demonstrating that PU.1 expression increased with the induction of osteoclastogenesis by either 1,25-dihydroxyvitamin D3 or dexamethasone. Consistent with the participation of PU.1 in osteoclastogenesis, we found that the development of both osteoclasts and macrophages is arrested in PU.1-deficient mice. Reflecting the absence of osteoclasts, PU.1-/- mice exhibit the classic hallmarks of osteopetrosis, a family of sclerotic bone diseases. These animals were rescued by marrow transplantation, with complete restoration of osteoclast and macrophage differentiation, verifying that the PU.1 lesion is intrinsic to haematopoietic cells. The absence of both osteoclasts and macrophages in PU.1-mutant animals suggests that the transcription factor regulates the initial stages of myeloid differentiation, and that its absence represents the earliest developmental osteopetrotic mutant yet described.
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.
Damage to neonatal and adult tissues always incites an influx of inflammatory neutrophils and macrophages. Besides clearing the wound of invading microbes, these cells are believed to be crucial ...coordinators of the repair process, acting both as professional phagocytes to clear wound debris and as a major source of wound growth factor signals. Here we report wound healing studies in the PU.1 null mouse, which is genetically incapable of raising the standard inflammatory response because it lacks macrophages and functioning neutrophils. Contrary to dogma, we show that these “macrophageless” mice are able to repair skin wounds with similar time course to wild-type siblings, and that repair appears scar-free as in the embryo, which also heals wounds without raising an inflammatory response. The growth factor and cytokine profile at the wound site is changed, cell death is reduced, and dying cells are instead engulfed by stand-in phagocytic fibroblasts. We also show that hyperinnervation of the wound site, previously believed to be a consequence of inflammation, is present in the PU.1 null wound, too.
Apoptosis is one of the key tools used by an embryo to regulate cell numbers and sculpt body shape. Although massive numbers of cells die during development, they are so rapidly phagocytosed that ...very few corpses are ever seen in most embryonic tissues. In this paper, we focus on the catastrophic cell death that occurs as the developing footplate is remodelled to transform webbed regions into free interdigital spaces. In the wild-type embryo, these dead cells are rapidly engulfed and cleared by macrophages. We show that in a macrophageless mouse embryo, null for the haemopoetic-lineage-specific transcription factor, PU.1, the task of phagocytosis is taken over by 'stand-in' mesenchymal neighbours in a clear example of cell redundancy. However, it takes three times as many of these mesenchymal phagocytes to complete the task and, at each stage of the clearance process - in the recognition of apoptotic debris, its engulfment and finally its digestion - they appear to be less efficient than macrophages. A molecular explanation for this may be that several of the engulfment genes expressed by macrophages, including the ABC1 transporter (believed to be part of the phagocytic machinery conserved from Caenorhabditis elegans to mouse), are not upregulated by these 'stand-in' phagocytes.
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 TGFalpha or the EGF receptor. Homozygous mutant cell lines respond mitogenically to TGFalpha, 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.
We have isolated a cDNA clone, PU.1, that codes for a new tissue-specific DNA binding protein. Analysis of the binding site by methylation interference and DNAase 1 protection revealed that the PU.1 ...protein recognized a purine-rich sequence, 5'-GAGGAA-3' (PU box). The PU.1 protein was shown to be a transcriptional activator that is expressed in macrophages and B cells. cDNA constructions used to generate proteins lacking portions of either the amino- or carboxy-terminal ends of the PU.1 protein placed the DNA binding domain in the highly basic carboxy-terminal domain of the protein. The amino acid sequence in the binding domain of PU.1 has considerable identity with proteins belonging to the ets oncogene family.
PU.1 recruits the binding of a second B cell-restricted nuclear factor, NF-EM5, to a DNA site in the immunoglobulin κ 3′ enhancer. DNA binding by NF-EM5 requires a protein-protein interaction with ...PU.1 and specific DNA contacts. Dephosphorylated PU.1 bound to DNA but did not interact with NF-EM5. Analysis of serine-to-alanine mutations in PU.1 indicated that serine 148 (Ser$^{148}$) is required for protein-protein interaction. PU.1 produced in bacteria did not interact with NF-EM5. Phosphorylation of bacterially produced PU.1 by purified casein kinase II modified it to a form that interacted with NF-EM5 and that recruited NF-EM5 to bind to DNA. Phosphopeptide analysis of bacterially produced PU.1 suggested that Ser$^{148}$ is phosphorylated by casein kinase II. This site is also phosphorylated in vivo. Expression of wild-type PU.1 increased expression of a reporter construct containing the PU.1 and NF-EM5 binding sites nearly sixfold, whereas the Ser$^{148}$ mutant form only weakly activated transcription. These results demonstrate that phosphorylation of PU.1 at Ser$^{148}$ is necessary for interaction with NF-EM5 and suggest that this phosphorylation can regulate transcriptional activity.
The ets family transcription factor PU.1 is expressed in monocytes/macrophages, neutrophils, mast cells, B cells, and early erythroblasts, but not in T cells. We have recently shown that PU.1 gene ...disruption results in mice with no detectable monocytes/macrophages and B cells but T-cell development is retained. Although neutrophil development occurred in these mice, it was delayed and markedly reduced. We now proceed to demonstrate that PU. 1 null hematopoietic cells fail to proliferate or form colonies in response to macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF), and granulocyte/macrophage CSF (GM-CSF). In contrast, PU.1 null cells did proliferate and form colonies in response to interleukin-3 (IL-3), although the response was reduced as compared with control littermates. Compared with control cells, PU.1 null cells had minimal expression of G- and GM-CSF receptors and no detectable M-CSF receptors. The size of individual myeloid colonies produced from PU.1 null primitive and committed myeloid progenitors in the presence of IL-3, IL-6, and stem cell factor (SCF) were reduced compared with controls. Under these conditions, PU.1 null progenitors produced neutrophils but not monocytes/macrophages. These observations suggest that PU.1 gene disruption induces additional cell-autonomous effects that are independent of the alterations in myeloid growth factor receptor expression. Our results demonstrate that PU.1 gene disruption affects a number of developmentally regulated hematopoietic processes that can, at least in part, explain the changes in myeloid development and reduction in myeloid and neutrophil expansion observed in PU.1 null mice.
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.
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