VHL: oxygen sensing and vasculogenesis.
Explore the source record for details and available documents.
Aryl Hydrocarbon Receptor Nuclear Translocator↗
Biomedical subjects
M C Simon
Publications and source records attributed to M C Simon.
Hypoxia, HIFs, and cardiovascular development.
Explore the source record for details and available documents.
Animals↗
Hypoxic gene regulation in differentiating ES cells.
Explore the source record for details and available documents.
Animals↗
Targeted mutation of the murine arylhydrocarbon receptor nuclear translocator 2 (Arnt2) gene reveals partial redundancy with Arnt.
The ubiquitously expressed basic helix-loop-helix (bHLH)-PAS protein ARNT (arylhydrocarbon receptor nuclear transporter) forms transcriptionally active heterodimers with a variety of other bHLH-PAS proteins, including HIF-1alpha (hypoxia-inducible factor-1alpha) and AHR (arylhydrocarbon receptor). These complexes regulate gene expression in response to hypoxia and xenobiotics, respectively, and mutation of the murine Arnt locus results in embryonic death by day 10.5 associated with placental, vascular, and hematopoietic defects. The closely related protein ARNT2 is highly expressed in the central nervous system and kidney and also forms complexes with HIF-1alpha and AHR. To assess unique roles for ARNT2 in development, and reveal potential functional overlap with ARNT, we generated a targeted null mutation of the murine Arnt2 locus. Arnt2(-/-) embryos die perinatally and exhibit impaired hypothalamic development, phenotypes previously observed for a targeted mutation in the murine bHLH-PAS gene Sim1 (Single-minded 1), and consistent with the recent proposal that ARNT2 and SIM1 form an essential heterodimer in vivo [Michaud, J. L., DeRossi, C., May, N. R., Holdener, B. C. & Fan, C. (2000) Mech. Dev. 90, 253-261]. In addition, cultured Arnt2(-/-) neurons display decreased hypoxic induction of HIF-1 target genes, demonstrating formally that ARNT2/HIF-1alpha complexes regulate oxygen-responsive genes. Finally, a strong genetic interaction between Arnt and Arnt2 mutations was observed, indicating that either gene can fulfill essential functions in a dose-dependent manner before embryonic day 8.5. These results demonstrate that Arnt and Arnt2 have both unique and overlapping essential functions in embryonic development.
Alleles↗
PU.1 exhibits partial functional redundancy with Spi-B, but not with Ets-1 or Elf-1.
Previously it was shown that the Ets proteins, PU.1 and Spi-B, exhibit functional redundancy in B lymphocytes. To investigate the possibility that PU.1 or Spi-B or both share overlapping roles with Ets-1 or Elf-1, PU.1(+/-)Ets-1(-/-), PU.1(+/-)Elf-1(-/-), and Spi-B(-/-)Ets-1(-/-) animals were generated. No blood cell defects were observed in these animals except those previously reported for Ets-1(-/-) mice. Therefore, no genetic overlap was detected between PU.1 or Spi-B with Ets-1 or Elf-1. In contrast, the results confirmed functional redundancy for PU.1 and Spi-B in that PU.1(+/-)Spi-B(-/-) bone marrow progenitors yielded smaller colonies in methylcellulose cultures than did wild-type, PU.1(+/-) or Spi-B(-/-) progenitors. In addition, PU.1(+/-)Spi-B(+/+), PU.1(+/-)Spi-B(+/-), and PU.1(+/-) Spi-B(-/-) mice displayed extramedullary splenic hematopoiesis. In summary, PU.1 and Spi-B regulate common target genes required for proliferation of hematopoietic progenitors or their committed descendants, whereas Ets-1 or Elf-1 do not appear to regulate shared target genes with PU.1 or Spi-B.
Animals↗
Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34(+) cells after ex vivo expansion.
OBJECTIVE: The relationship between phenotype and function in ex vivo-cultured human hematopoietic stem cells (HSC) remains poorly understood. We investigated the effects of a short-term serum-free culture on the relationship between stem cell phenotype, cell division history, and function in human CD34(+) cells. METHODS: G-CSF-mobilized peripheral CD34(+) cells were cultured for 4 days with stem cell factor, flt-3 ligand, and thrombopoietin. The phenotype (CD34, CD38, HLA-DR, c-kit), cell division history, colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and NOD/SCID repopulating activities were evaluated at Day 0 and 4. RESULTS: We observed a loss of CD38, HLA-DR, and c-kit surface expression resulting in a drastic increase in CD34(+)CD38(-), CD34(+)HLA-DR(-), and CD34(+)c-kit(-/low) cells at Day 4. In contrast, the frequency of Thy-1(+) cells was maintained. We observed a 1.3-fold expansion of CFC, a 4.8-fold increase in LTC-IC, and an overall maintenance of the NOD/SCID repopulating cell activity. CD34(+)CD38(-) and CD34(+)HLA-DR(-) cells detected at Day 4 displayed the most active pattern of division (4 to 5 divisions) whereas 60% of CD34(+)Thy-1(+) cells divided 0 to 2 times during the same period. At Day 4, the NOD/SCID repopulating activity was associated with Thy-1(+) cells with no more than 2 divisions. CONCLUSIONS: Our results show that the relationship between stem cell phenotype and function is dramatically altered in cultured CD34(+) cells. Thy-1 expression and cell division history appear to be superior to CD38, HLA-DR, and c-kit, or to homing molecules (CXCR4, VLA-4) as predictors of the repopulating activity of cultured peripheral CD34(+) cells.
ADP-ribosyl Cyclase↗
PU.1/Spi-B regulation of c-rel is essential for mature B cell survival.
PU.1(+/-)Spi-B(-/-) mice exhibit reduced numbers of immature and mature B lymphocytes, which exhibit severe defects in response to BCR-mediated stimulation and poor survival. We found that expression of c-rel, a member of the Rel/NF-kappa B family, is dramatically reduced in PU.1(+/-)Spi-B(-/-) splenic B cells. Analysis of the murine c-rel promoter identified three PU.1/Spi-B binding sites critical for c-rel promoter activity. Furthermore, reintroduction of Rel protein restored wild-type B cell numbers to mice reconstituted with PU.1(+/-)Spi-B(-/-) bone marrow. These findings are the first to demonstrate that a member of the Rel/NF-kappa B family is directly regulated by Ets proteins and dissect the molecular basis for the function of two Ets factors, PU.1 and Spi-B, in promoting B lymphocyte survival.
Animals↗
Hypoxia-inducible factor and the development of stem cells of the cardiovascular system.
Decreased oxygen (O2) levels activate hypoxia-inducible factor (HIF-1) to induce genes involved in glycolysis, glucose transport, erythropoiesis, and angiogenesis. Mutations in various HIF-1 subunits have contributed to our understanding of the role hypoxia plays during early embryonic development in general and the cardiovascular system in particular. We propose that HIF-1 is important for the generation, proliferation, maintenance, and differentiation of the early cardiovascular system. Understanding aberrations in these hypoxic responses is important since they contribute to serious human disease such as ischemia and tumorigenesis. In this review we will focus on the critical role of O2 in regulating cardiovascular events during early embryonic development.
Animals↗
Mop3 is an essential component of the master circadian pacemaker in mammals.
Circadian oscillations in mammalian physiology and behavior are regulated by an endogenous biological clock. Here we show that loss of the PAS protein MOP3 (also known as BMAL1) in mice results in immediate and complete loss of circadian rhythmicity in constant darkness. Additionally, locomotor activity in light-dark (LD) cycles is impaired and activity levels are reduced in Mop3-/- mice. Analysis of Period gene expression in the suprachiasmatic nucleus (SCN) indicates that these behavioral phenotypes arise from loss of circadian function at the molecular level. These results provide genetic evidence that MOP3 is the bona fide heterodimeric partner of mCLOCK. Furthermore, these data demonstrate that MOP3 is a nonredundant and essential component of the circadian pacemaker in mammals.
ARNTL Transcription Factors↗
Placental cell fates are regulated in vivo by HIF-mediated hypoxia responses.
Placental development is profoundly influenced by oxygen (O(2)) tension. Human cytotrophoblasts proliferate in vitro under low O(2) conditions but differentiate at higher O(2) levels, mimicking the developmental transition they undergo as they invade the placental bed to establish the maternal-fetal circulation in vivo. Hypoxia-inducible factor-1 (HIF-1), consisting of HIF-1alpha and ARNT subunits, activates many genes involved in the cellular and organismal response to O(2) deprivation. Analysis of Arnt(-/-) placentas reveals an aberrant cellular architecture due to altered cell fate determination of Arnt(-/-) trophoblasts. Specifically, Arnt(-/-) placentas show greatly reduced labyrinthine and spongiotrophoblast layers, and increased numbers of giant cells. We further show that hypoxia promotes the in vitro differentiation of trophoblast stem cells into spongiotrophoblasts as opposed to giant cells. Our results clearly establish that O(2) levels regulate cell fate determination in vivo and that HIF is essential for mammalian placentation. The unique placental phenotype of Arnt(-/-) animals also provides an important tool for studying the disease of preeclampsia. Interestingly, aggregation of Arnt(-/-) embryonic stem (ES) cells with tetraploid wild-type embryos rescues their placental defects; however, these embryos still die from yolk sac vascular and cardiac defects.
Animals↗
A minimal c-fes cassette directs myeloid-specific expression in transgenic mice.
The c-fes proto-oncogene encodes a 92-kd protein tyrosine kinase whose expression is restricted largely to myeloid and endothelial cells in adult mammals. A 13.2-kilobase (kb) human c-fes genomic fragment was previously shown to contain cis-acting element(s) sufficient for a locus control function in bone marrow macrophages. Locus control regions (LCRs) confer transgene expression in mice that is integration site independent, copy number dependent, and similar to endogenous murine messenger RNA levels. To identify sequences required for this LCR, c-fes transgenes were analyzed in mice. Myeloid-cell-specific, deoxyribonuclease-I-hypersensitive sites localized to the 3' boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to endogenous c-fes, independent of integration site. We define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to intron 3 of the human locus. When this 2.5-kb DNA fragment was linked to a c-fes complementary DNA regulated by its own 446-base-pair promoter, integration-site-independent, copy-number-dependent transcription was observed in myeloid cells in transgenic mice. Furthermore, this 2.5-kb cassette directed expression of a heterologous gene (enhanced green fluorescent protein) exclusively in myeloid cells. The c-fes regulatory unit represents a novel reagent for targeting gene expression to macrophages and neutrophils in transgenic mice.
Animals↗
The role of ARNT2 in tumor angiogenesis and the neural response to hypoxia.
The Hypoxia-Inducible Factor-1 (HIF-1) activates the transcription of many genes required for cellular and organismal responses to oxygen deprivation. The HIF-1 complex is composed of the ubiquitously expressed basic helix-loop-helix/PAS (bHLH/PAS) proteins HIF-1alpha and Arylhydrocarbon Receptor Nuclear Translocator (ARNT). ARNT2 is a conserved ARNT homolog that is highly expressed in neurons, suggesting that ARNT2/HIF-1alpha heterodimers mediate transcriptional responses to oxygen deprivation in the nervous system. We show here that ARNT2 forms functional HIF complexes in vivo, and that ARNT2 restores hypoxia-induced gene expression to ARNT-deficient ES cells and hepatocytes. Formation of neural ARNT2/HIF-1alpha complexes in Arnt(-/-) ES cell-derived teratocarcinomas may explain why these tumors express VEGF, vascularize and grow efficiently, in contrast to ARNT-deficient hepatomas. Interestingly, all neural cell types studied accumulate both ARNT- and ARNT2-containing HIF complexes. We conclude that ARNT2 forms functional HIF complexes in neurons and plays an integral role in hypoxic responses in the CNS.
Animals↗
HIF-1 is essential for multilineage hematopoiesis in the embryo.
Explore the source record for details and available documents.
Animals↗
Abnormal Stat activation, hematopoietic homeostasis, and innate immunity in c-fes-/- mice.
The c-fes protooncogene encodes a nonreceptor tyrosine kinase (Fes) implicated in cytokine receptor signal transduction, neutrophil survival, and myeloid differentiation. To determine the role of Fes in embryonic development and hematopoiesis, we engineered a null mutation of the murine c-fes locus. c-fes-/- mice are viable but not born in the expected Mendelian ratios. Live born c-fes-/- mice exhibit lymphoid/myeloid homeostasis defects, compromised innate immunity, and increased Stat activation in response to GM-CSF and IL-6 signaling. Therefore, increased cytokine responsiveness in the absence of Fes leads to abnormal myeloid proliferation and functional defects in the macrophage lineage.
Animals↗
The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues.
Proapoptotic Bcl-2 family members have been proposed to play a central role in regulating apoptosis. However, mice lacking bax display limited phenotypic abnormalities. As presented here, bak(-/-) mice were found to be developmentally normal and reproductively fit and failed to develop any age-related disorders. However, when Bak-deficient mice were mated to Bax-deficient mice to create mice lacking both genes, the majority of bax(-/-)bak(-/-) animals died perinatally with fewer than 10% surviving into adulthood. bax(-/-)bak(-/-) mice displayed multiple developmental defects, including persistence of interdigital webs, an imperforate vaginal canal, and accumulation of excess cells within both the central nervous and hematopoietic systems. Thus, Bax and Bak have overlapping roles in the regulation of apoptosis during mammalian development and tissue homeostasis.
Abnormalities, Multiple↗
The Ets factors PU.1 and Spi-B regulate the transcription in vivo of P2Y10, a lymphoid restricted heptahelical receptor.
To investigate the in vivo functions of PU.1 and Spi-B, two highly related Ets transcription factors, we previously generated PU. 1(+/+)Spi-B(-/-) and PU.1(+/-)Spi-B(-/-) mice and demonstrated a significant decrease in B-cell receptor (BCR) signaling in mutants. Major components of BCR signaling appear to be expressed at normal levels in these mice, implying that PU.1 and Spi-B cooperate in the transcription of additional target genes important for antigen receptor signaling. We used subtractive hybridization to identify novel in vivo PU.1/Spi-B target genes and determined that the expression of a heptahelical receptor, P2Y10, is dramatically reduced in PU.1(+/-)Spi-B(-/-) B-cells. Further analysis shows that P2Y10 expression is restricted to lymphoid cells and parallels that of Spi-B in B-lymphocytes. Lastly, the P2Y10 promoter contains a PU. 1/Spi-B binding site functionally required for efficient transcription in B-cells. Thus, P2Y10 is likely to be a direct in vivo transcriptional target for PU.1 and Spi-B and provides a unique model to explore transcriptional regulation by this Ets factor subfamily. Furthermore, P2Y10 suggests an intriguing connection between heterotrimeric G-proteins and BCR signaling.
3T3 Cells↗
Multilineage embryonic hematopoiesis requires hypoxic ARNT activity.
Although most cells undergo growth arrest during hypoxia, endothelial cells and placental cytotrophoblasts proliferate in response to low O(2). We demonstrate that proliferation of embryonic multilineage hematopoietic progenitors is also regulated by a hypoxia-mediated signaling pathway. This pathway requires HIF-1 (HIF-1alpha/ARNT heterodimers) because Arnt(-/-) embryoid bodies fail to exhibit hypoxia-mediated progenitor proliferation. Furthermore, Arnt(-/-) embryos exhibit decreased numbers of yolk sac hematopoietic progenitors. This defect is cell extrinsic, is accompanied by a decrease in ARNT-dependent VEGF expression, and is rescued by exogenous VEGF. Therefore, "physiologic hypoxia" encountered by embryos is essential for the proliferation or survival of hematopoietic precursors during development.
Animals↗
SPI-B activates transcription via a unique proline, serine, and threonine domain and exhibits DNA binding affinity differences from PU.1.
SPI-B is a B lymphocyte-specific Ets transcription factor that shares a high degree of similarity with PU.1/SPI-1. In direct contrast to PU.1(-/-) mice that die in utero and lack monocytes, neutrophils, B cells, and T cells, Spi-B-/- mice are viable and exhibit a severe B cell proliferation defect. Since PU.1 is expressed at wild type levels in Spi-B-/- B cells, the mutant mice provide genetic evidence that SPI-B and PU.1 have at least some non-redundant roles in B lymphocytes. To begin to understand the molecular basis for these defects, we delineated functional domains of SPI-B for comparison to those of PU.1. By using a heterologous co-transfection system, we identified two independent transactivation domains in the N terminus of SPI-B. Interestingly, only one of these domains (amino acids 31-61), a proline/serine/threonine-rich region, unique among Ets proteins, is necessary for transactivation of the immunoglobulin lambda light chain enhancer. This transactivation motif is in marked contrast to PU.1, which contains acidic and glutamine-rich domains. In addition, we describe a functional PU.1 site within the c-FES promoter which SPI-B fails to bind efficiently and transactivate. Finally, we show that SPI-B interacts with the PU.1 cofactors Pip, TBP, c-Jun and with lower affinity to nuclear factor interleukin-6beta and retinoblastoma. Taken together, these data suggest that SPI-B binds DNA with a different affinity for certain sites than PU.1 and harbors different transactivation domains. We conclude that SPI-B may activate unique target genes in B lymphocytes and interact with unique, although currently unidentified, cofactors.
Amino Acid Sequence↗