Biomedical subjects

Osamu Ohneda

Publications and source records attributed to Osamu Ohneda.

18 recordsLinked to original sources

PubMed2006

Characterization of GATA-1(+) hemangioblastic cells in the mouse embryo.

Hemangioblasts are thought to be one of the sources of hematopoietic progenitors, yet little is known about their localization and fate in the mouse embryo. We show here that a subset of cells co-expressing the hematopoietic marker GATA-1 and the endothelial marker VE-cadherin localize on the yolk sac blood islands at embryonic day 7.5. Clonal analysis demonstrated that GATA-1(+) cells isolated from E7.0-7.5 embryos include a common precursor for hematopoietic and endothelial cells. Moreover, this precursor possesses primitive and definitive hematopoietic bipotential. By using a transgenic complementation rescue approach, GATA-1(+) cell-derived progenitors were selectively restored in Runx1-deficient mice. In the rescued mice, definitive erythropoiesis was recovered but the rescued progenitors did not display multilineage hematopoiesis or intra-aortic hematopoietic clusters. These results provide evidence of the presence of GATA-1(+) hemangioblastic cells in the extra-embryonic region and also their functional contribution to hematopoiesis in the embryo.

Animals↗

PubMed2006

GATA-4 incompletely substitutes for GATA-1 in promoting both primitive and definitive erythropoiesis in vivo.

Vertebrate GATA transcription factors have been classified into two subgroups; GATA-1, GATA-2, and GATA-3 are expressed in hematopoietic cells, whereas GATA-4, GATA-5, and GATA-6 are expressed in mesoendoderm-derived tissues. We previously discovered that expression of GATA-2 or GATA-3 under the transcriptional control for the Gata1 gene eliminates lethal anemia in Gata1 germ line mutant mice (Gata1.05/Y). Here, we show that the GATA-4 expression by the same regulatory cassette prolongs the life span of Gata1.05/Y embryos from embryonic day 12.5 to 15.5 but fails to abrogate its embryonic lethality. Gata1.05/Y mice bearing the GATA-4 transgene showed impaired maturation of both primitive and definitive erythroid cells and defective erythroid cell expansion in fetal liver. Moreover, the incidence of apoptosis was observed prominently in primitive erythroid cells. In contrast, a GATA-4-GATA-1 chimeric protein prepared by linking the N-terminal region of GATA-4 to the C-terminal region of GATA-1 significantly promoted the differentiation and survival of primitive erythroid cells, although this protein is still insufficient for rescuing Gata1.05/Y embryos from lethal anemia. These data thus show a functional incompatibility between hematopoietic and endodermal GATA factors in vivo and provide evidence indicating specific roles of the C-terminal region of GATA-1 in primitive erythropoiesis.

Amino Acid Sequence↗

PubMed2006

Transgene insertion in proximity to the c-myb gene disrupts erythroid-megakaryocytic lineage bifurcation.

The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival, and differentiation of hematopoietic cells. We established three lines of erythropoietin receptor-transgenic mice and found that one of them exhibited anemia, thrombocythemia, and splenomegaly. These abnormalities were independent of the function of the transgenic erythropoietin receptor and were observed exclusively in mice harboring the transgene homozygously, suggesting transgenic disruption of a certain gene. The transgene was inserted 77 kb upstream of the c-myb gene, and c-Myb expression was markedly decreased in megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs) of the homozygous mutant mice. In the bone marrows and spleens of the mutant mice, numbers of megakaryocytes were increased and numbers of erythroid progenitors were decreased. These abnormalities were reproducible in vitro in a coculture assay of MEPs with OP9 cells but eliminated by the retroviral expression of c-Myb in MEPs. The erythroid/megakaryocytic abnormalities were reconstituted in mice in vivo by transplantation of mutant mouse bone marrow cells. These results demonstrate that the transgene insertion into the c-myb gene far upstream regulatory region affects the gene expression at the stage of MEPs, leading to an imbalance between erythroid and megakaryocytic cells, and suggest that c-Myb is an essential regulator of the erythroid-megakaryocytic lineage bifurcation.

Animals↗

PubMed2006

Real-time monitoring of stress erythropoiesis in vivo using Gata1 and beta-globin LCR luciferase transgenic mice.

Erythroid progenitors have the potential to proliferate rapidly in response to environmental stimuli. This process is referred to as stress erythropoiesis, with erythropoietin (EPO) playing central roles in its promotion. In this study, we wanted to elucidate the molecular mechanisms governing the regulation of stress erythropoiesis and the maintenance of red-cell homeostasis. This was achieved by our development of a noninvasive real-time monitoring system for erythropoiesis using transgenic mouse lines expressing luciferase under the control of the mouse Gata1 hematopoietic regulatory domain (G1-HRD-luc) or human beta-globin locus control region (Hbb-LCR-luc). Optical bioluminescence images revealed that the luciferase was specifically expressed in spleen and bone marrow and was induced rapidly in response to anemia and hypoxia stimuli. The G1-HRD-luc activity tracked the emergence and disappearance of proerythroblast-stage progenitors, whereas the Hbb-LCR-luc activity tracked erythroblasts and later stage erythroid cells. Increased plasma EPO concentration preceded an increase in G1-HRD-luc, supporting our contention that EPO acts as the key upstream signal in stress erythropoiesis. Hence, we conclude that G1-HRD-luc and Hbb-LCR-luc reporters are differentially activated during stress erythropoiesis and that the transgenic mouse lines used serve as an important means for understanding the homeostatic regulation of erythropoiesis.

Animals↗

PubMed2006

Combinatorial Gata2 and Sca1 expression defines hematopoietic stem cells in the bone marrow niche.

The interaction between stem cells and their supportive microenvironment is critical for their maintenance, function, and survival. Whereas hematopoietic stem cells (HSCs) are among the best characterized of tissue stem cells, their precise site of residence (referred to as the niche) in the adult bone marrow has not been precisely defined. In this study, we found that a Gata2 promoter directs activity in all HSCs. We show that HSCs can be isolated efficiently from bone marrow cells by following Gata2-directed GFP fluorescence, and that they can also be monitored in vivo. Each individual GFP-positive cell lay in a G0/G1 cell cycle state, in intimate contact with osteoblasts beside the endosteum, at the edge of the bone marrow. We conclude that the HSC niche is composed of solitary cells and that adult bone marrow HSC are not clustered.

Animals↗

PubMed2005

Enhanced erythropoiesis mediated by activation of the renin-angiotensin system via angiotensin II type 1a receptor.

Although clinical and experimental studies have long suggested a role for the renin-angiotensin system (RAS) in the regulation of erythropoiesis, the molecular basis of this role has not been well understood. We report here that transgenic mice carrying both the human renin and human angiotensinogen genes displayed persistent erythrocytosis as well as hypertension. To identify the receptor molecule responsible for this phenotype, we introduced both transgenes into the AT1a receptor null background and found that the hematocrit level in the compound mice was restored to the normal level. Angiotensin II has been shown to influence erythropoiesis by two means, up-regulation of erythropoietin levels and direct stimulation of erythroid progenitor cells. Thus, we conducted bone marrow transplantation experiments and clarified that AT1a receptors on bone marrow-derived cells were dispensable for RAS-dependent erythrocytosis. Plasma erythropoietin levels and kidney erythropoietin mRNA expression in the double transgenic mice were significantly increased compared with those of the wild-type control, while the elevated plasma erythropoietin levels were significantly attenuated in the compound mice. These results provide clear genetic evidence that activated RAS enhances erythropoiesis through the AT1a receptor of kidney cells and that this effect is mediated by the elevation of plasma erythropoietin levels in vivo.

Angiotensin II↗

PubMed2005

Graded levels of GATA-1 expression modulate survival, proliferation, and differentiation of erythroid progenitors.

Transcription factor GATA-1 plays an important role in gene regulation during the development of erythroid cells. Several reports suggest that GATA-1 plays multiple roles in survival, proliferation, and differentiation of erythroid cells. However, little is known about the relationship between the level of GATA-1 expression and its nature of multifunction to affect erythroid cell fate. To address this issue, we developed in vitro embryonic stem (ES) culture system by using OP9 stromal cells (OP9/ES cell co-culture system), and cultured the mutant (GATA-1.05 and GATA-1-null) and wild type (WT)ES cells, respectively. By using this OP9/ES cell co-culture system, primitive and definitive erythroid cells were developed individually, and we examined how expression level of GATA-1 affects the development of erythroid cells. GATA-1.05 ES-derived definitive erythroid cells were immature with the appearance of proerythroblasts, and highly proliferated, compared with WT and GATA-1-null ES-derived erythroid cells. Extensive studies of cell cycle kinetics revealed that the GATA-1.05 proerythroblasts accumulated in S phase and expressed lower levels of p16(INK4A) than WT ES cell-derived proerythroblasts. We concluded that GATA-1 must achieve a critical threshold activity to achieve selective activation of specific target genes, thereby influencing the developmental decision of an erythroid progenitor cell to undergo apoptosis, proliferation, or terminal differentiation.

Animals↗

PubMed2005

Transgenic over-expression of GATA-1 mutant lacking N-finger domain causes hemolytic syndrome in mouse erythroid cells.

Transcription factor GATA-1 is essential for erythroid cell differentiation. GATA-binding motifs have been found in the regulatory regions of various erythroid-specific genes, suggesting that GATA-1 contributes to gene regulation during the entire process of erythropoiesis. A GATA-1 germ-line mutation results in embryonic lethality due to defective primitive erythropoiesis and GATA-1-null embryonic stem cells fails to differentiate beyond the proerythroblast stage. Therefore, the precise roles of GATA-1 in the later stages of erythropoiesis could not be clarified. Under the control of a GATA-1 gene hematopoietic regulatory domain, a GATA-1 mutant lacking the N-finger domain (DeltaNF mutant) was over-expressed in mice. These mice exhibited abnormal morphology in peripheral red blood cells (RBCs), reticulocytosis, splenomegaly, and erythroid hyperplasia, indicating compensated hemolysis. These mice were extremely sensitive to phenylhydrazine (PHZ), an agent that induces hemolysis, and their RBCs were osmotically fragile. Importantly, the hemolytic response to PHZ was partially restored by the simultaneous expression of wild-type GATA-1 with the DeltaNF mutant, supporting our contention that DeltaNF protein competitively inhibits the function of endogenous GATA-1. These data provide the first in vivo evidence that the NF domain contributes to the gene regulation that is critical for differentiation and survival of mature RBCs in postnatal erythropoiesis.

Animals↗

PubMed2005

GATA motifs regulate early hematopoietic lineage-specific expression of the Gata2 gene.

Transcription factor GATA-2 is essential for definitive hematopoiesis, which developmentally emerges from the para-aortic splanchnopleura (P-Sp). The expression of a green fluorescent protein (GFP) reporter placed under the control of a 3.1-kbp Gata2 gene regulatory domain 5' to the distal first exon (IS) mirrored that of the endogenous Gata2 gene within the P-Sp and yolk sac (YS) blood islands of embryonic day (E) 9.5 murine embryos. The P-Sp- and YS-derived GFP(+) fraction of flow-sorted cells dissociated from E9.5 transgenic embryos contained far more CD34(+)/c-Kit(+) cells than the GFP(-) fraction did. When cultured in vitro, the P-Sp GFP(+) cells generated both immature hematopoietic and endothelial cell clusters. Detailed transgenic mouse reporter expression analyses demonstrate that five GATA motifs within the 3.1-kbp Gata2 early hematopoietic regulatory domain (G2-EHRD) were essential for GFP expression within the dorsal aortic wall, where hemangioblasts, the earliest precursors possessing both hematopoietic and vascular developmental potential, are thought to reside. These results thus show that the Gata2 gene IS promoter is regulated by a GATA factor(s) and selectively marks putative hematopoietic/endothelial precursor cells within the P-Sp.

Amino Acid Motifs↗

PubMed2005

Molecular determinants of NOTCH4 transcription in vascular endothelium.

The process whereby the primitive vascular network develops into the mature vasculature, known as angiogenic vascular remodeling, is controlled by the Notch signaling pathway. Of the two mammalian Notch receptors expressed in vascular endothelium, Notch1 is broadly expressed in diverse cell types, whereas Notch4 is preferentially expressed in endothelial cells. As mechanisms that confer Notch4 expression were unknown, we investigated how NOTCH4 transcription is regulated in human endothelial cells and in transgenic mice. The NOTCH4 promoter and the 5' portion of NOTCH4 assembled into an endothelial cell-specific histone modification pattern. Analysis of NOTCH4 primary transcripts in human umbilical vein endothelial cells by RNA fluorescence in situ hybridization revealed that 36% of the cells transcribed one or both NOTCH4 alleles. The NOTCH4 promoter was sufficient to confer endothelial cell-specific transcription in transfection assays, but intron 1 or upstream sequences were required for expression in the vasculature of transgenic mouse embryos. Cell-type-specific activator protein 1 (AP-1) complexes occupied NOTCH4 chromatin and conferred endothelial cell-specific transcription. Vascular angiogenic factors activated AP-1 and reprogrammed the endogenous NOTCH4 gene in HeLa cells from a repressed to a transcriptionally active state. These results reveal an AP-1-Notch4 pathway, which we propose to be crucial for transducing angiogenic signals and to be deregulated upon aberrant signal transduction in cancer.

5' Flanking Region↗

PubMed2004

Essential role of synoviolin in embryogenesis.

We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect. For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno(-/-)) mice by genetargeted disruption. Strikingly, all fetuses lacking syno died in utero around embryonic day 13.5, although Hrd1p, a yeast orthologue of Synoviolin, is non-essential for survival. Histologically, hypocellularity and aberrant apoptosis were noted in the syno(-/-) fetal liver. Moreover, definitive erythropoiesis was affected in non-cell autonomous manner in syno(-/-) embryos, causing death in utero. Cultured embryonic fibroblasts derived from syno(-/-) mice were more susceptible to endoplasmic reticulum stress-induced apoptosis than those from syno(+/+) mice, but the susceptibility was rescued by overexpression of synoviolin. Our findings emphasized the indispensable role of the Synoviolin in embryogenesis.

Adenoviridae↗

PubMed2004

Leukemogenesis caused by incapacitated GATA-1 function.

GATA-1 is essential for the development of erythroid and megakaryocytic lineages. We found that GATA-1 gene knockdown female (GATA-1.05/X) mice frequently develop a hematopoietic disorder resembling myelodysplastic syndrome that is characterized by the accumulation of progenitors expressing low levels of GATA-1. In this study, we demonstrate that GATA-1.05/X mice suffer from two distinct types of acute leukemia, an early-onset c-Kit-positive nonlymphoid leukemia and a late-onset B-lymphocytic leukemia. Since GATA-1 is an X chromosome gene, two types of hematopoietic cells reside within heterozygous GATA-1 knockdown mice, bearing either an active wild-type GATA-1 allele or an active mutant GATA-1.05 allele. In the hematopoietic progenitors with the latter allele, low-level GATA-1 expression is sufficient to support survival and proliferation but not differentiation, leading to the accumulation of progenitors that are easily targeted by oncogenic stimuli. Since such leukemia has not been observed in GATA-1-null/X mutant mice, we conclude that the residual GATA-1 activity in the knockdown mice contributes to the development of the malignancy. This de novo model recapitulates the acute crisis found in preleukemic conditions in humans.

Animals↗

PubMed2003

Identification and characterization of 2 types of erythroid progenitors that express GATA-1 at distinct levels.

Transcription factor GATA-1 is essential for the development of the erythroid lineage. To ascertain whether strict control of GATA-1 expression level is necessary for achieving proper erythropoiesis, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of the GATA-1 gene hematopoietic regulatory domain. We examined the GATA-1 expression level by exploiting the transgenic mice and found 2 GFP-positive hematopoietic progenitor fractions in the bone marrow. One is the GFPhigh fraction containing mainly CFU-E and proerythroblasts, which coexpress transferrin receptor, while the other is the GFPlow/transferrin receptor-negative fraction containing BFU-E. Since the intensity of green fluorescence correlates well with the expression level of GATA-1, these results indicate that GATA-1 is highly expressed in erythroid colony-forming unit (CFU-E) but low in erythroid burst-forming unit (BFU-E), suggesting that the incremental expression of GATA-1 is required for the formation of erythroid progenitors. We also examined GFP-positive fractions in the transgenic mouse spleen and fetal liver and identified fractions containing BFU-E and CFU-E, respectively. This study also presents an efficient method for enriching the CFU-E and BFU-E from mouse hematopoietic tissues.

Animals↗

PubMed2003

Frequent mutations in the GATA-1 gene in the transient myeloproliferative disorder of Down syndrome.

Transient myeloproliferative disorder (TMD) is a leukemoid reaction occurring occasionally in Down syndrome newborn infants. Acute megakaryocytic leukemia (AMKL) develops in approximately 20% to 30% of the cases with TMD. Recently, acquired mutations in the N-terminal activation domain of the GATA-1 gene, encoding the erythroid/megakaryocytic transcription factor GATA-1, have been reported in Down syndrome-related AMKL (DS-AMKL). To understand the multistep leukemogenesis in Down syndrome, GATA-1 mutations were investigated in patients with TMD. We show here that mutations in the GATA-1 gene were detected in 21 of 22 cases with TMD. Most of the mutations in TMD were located in the regions including exon 2 and were essentially identical to those observed in DS-AMKL. In the DS-AMKL cell line, MGS, which itself expresses only a truncated mutant of GATA-1, expression of full-length GATA-1 induced the differentiation toward the erythroid lineage. However, expression of the short form of GATA-1 did not induce erythroid differentiation. These results indicate that expression of GATA-1 with a defective N-terminal activation domain contributes to the expansion of TMD blast cells and that other genetic changes contribute to the development of AMKL in Down syndrome.

Age Factors↗

PubMed2003

Identification and characterization of stem cells in prepubertal spermatogenesis in mice.

The stem cell properties of gonocytes and prospermatogonia at prepubertal stages are still largely unknown: it is not clear whether gonocytes and prospermatogonia are a special cell type or similar to adult undifferentiated spermatogonia. To characterize these cells, we have established transgenic mice carrying EGFP (enhanced green fluorescence protein) cDNA under control of an Oct4 18-kb genomic fragment containing the minimal promoter and proximal and distal enhancers; Oct4 is reported to be expressed in undifferentiated spermatogonia at prepubertal stages. Generation of transgenic mice enabled us to purify gonocytes and prospermatogonia from the somatic cells of the testis. Transplantation studies of testicular cells so far have been done with a mixture of germ cells and somatic cells. This is the first report that establishes how to purify germ cells from total testicular cells, enabling evaluation of cell-autonomous repopulating activity of a subpopulation of prospermatogonia. We show that prospermatogonia differ markedly from adult spermatogonia in both the size of the KIT-negative population and cell cycle characteristics. The GFP(+) KIT(-) fraction of prospermatogonia has much higher repopulating activity than does the GFP(+)KIT(+) population in the adult environment. Interestingly, the GFP(+)KIT(+) population still exhibits repopulating activity, unlike adult KIT-positive spermatogonia. We also show that ALCAM, activated leukocyte cell adhesion molecule, is expressed transiently in gonocytes. Sertoli cells and myoid cells also express ALCAM at the same stage, suggesting that ALCAM may contribute to gonocyte-Sertoli cell adhesion and migration of gonoyctes toward the basement membrane.

Activated-Leukocyte Cell Adhesion Molecule↗

PubMed2003

HLF/HIF-2alpha is a key factor in retinopathy of prematurity in association with erythropoietin.

An HLF (HIF-1alpha-like factor)/HIF-2alpha-knockout mouse is embryonic lethal, preventing investigation of HLF function in adult mice. To investigate the role of HLF in adult pathological angiogenesis, we generated HLF-knockdown (HLF(kd/kd)) mice by inserting a neomycin gene sandwiched between two loxP sequences into exon 1 of the HLF gene. HLF(kd/kd) mice expressing 80-20% reduction, depending on the tissue, in wild-type HLF mRNA were fertile and apparently normal. Hyperoxia-normoxia treatment, used as a murine model of retinopathy of prematurity (ROP), induced neovascularization in wild-type mice, but not in HLF(kd/kd) mice, whereas prolonged normoxia following hyperoxic treatment caused degeneration of retinal neural layers in HLF(kd/kd) mice due to poor vascularization. Cre-mediated removal of the inserted gene recovered normal HLF expression and retinal neovascularization in HLF(kd/kd) mice. Expression levels of various angiogenic factors revealed that only erythropoietin (Epo) gene expression was significantly affected, in parallel with HLF expression. Together with the results from intraperitoneal injection of Epo into HLF(kd/kd) mouse, this suggests that Epo is one of the target genes of HLF responsible for experimental ROP.

Animals↗

PubMed2002

Erythroid-specific expression of the erythropoietin receptor rescued its null mutant mice from lethality.

Erythropoietin (Epo) and its receptor (EpoR) are indispensable to erythropoiesis. Although roles besides angiogenesis, such as neuroprotection and heart development, have been reported for the Epo-EpoR system, the precise contribution of Epo-EpoR to these nonhematopoietic tissues requires clarification. Exploiting a GATA-1 minigene cassette with hematopoietic regulatory domains, we established 2 lines of transgene-rescued EpoR-null mutant mice expressing EpoR exclusively in the hematopoietic lineage. Surprisingly, despite the lack of EpoR expression in nonhematopoietic tissues, these mice develop normally and are fertile. As such, we could exploit them for analyzing the roles of the Epo-EpoR system in adult hematopoiesis and in nonhematopoietic tissues. These rescued lines showed a differential level of EpoR expression in erythroid cells; one expressed approximately 40%, and the other expressed 120% of the wild-type EpoR level. A colony formation assay showed that erythroid progenitors in the 2 mutant lines exhibit distinct sensitivity to Epo. The circulating Epo level was much higher in the transgenic line with a lower EpoR expression. In response to induced anemia, the plasma Epo concentrations increased in both lines. Notably, the timing of the peak of plasma Epo concentration was delayed in both lines of rescued mice compared with wild type, suggesting that, in wild-type mice, nonhematopoietic EpoR contributes to the regulation of plasma Epo concentration. We thus conclude that nonhematopoietic expression of EpoR is dispensable to normal mouse development and that the expression level of EpoR regulates erythropoiesis by controlling the sensitivity of erythroid progenitors to Epo.

Anemia↗

PubMed2002

Mesenchymal stem cells in perichondrium express activated leukocyte cell adhesion molecule and participate in bone marrow formation.

Perichondrium in fetal limb is composed of undifferentiated mesenchymal cells. However, the multipotency of cells in this region and the role of perichondrium in bone marrow formation are not well understood. In this report, we purified and characterized perichondrial cells using a monoclonal antibody against activated leukocyte cell adhesion molecule (ALCAM) and investigated the role of perichondrial cells in hematopoietic bone marrow formation. ALCAM is expressed on hematopoietic cells, endothelial cells, bone marrow stromal cells, and mesenchymal stem cells and mediates homophilic (ALCAM-ALCAM)/heterophilic (ALCAM-CD6) cell adhesion. Here we show by immunohistochemical staining that ALCAM is expressed in perichondrium. ALCAM+ perichondrial cells isolated by FACS exhibit the characteristics of mesenchymal stem cells. ALCAM+ cells can differentiate into osteoblasts, adipocytes, chondrocytes, and stromal cells, which can support osteoclastogenesis, hematopoiesis, and angiogenesis. Furthermore, the addition of ALCAM-Fc or CD6-Fc to the metatarsal culture, the invasion of the blood vessels to a cartilage was inhibited. Our findings indicate that ALCAM+ perichondrial cells participate in vascular invasion by recruiting osteoclasts and vessels. These findings suggest that perichondrium might serve as a stem cell reservoir and play an important role in the early development of a bone and bone marrow.

Activated-Leukocyte Cell Adhesion Molecule↗