scholarly journals Infection-Responsive Expansion of the Hematopoietic Stem and Progenitor Cell Compartment in Zebrafish Is Dependent upon Inducible Nitric Oxide

2012 ◽  
Vol 10 (2) ◽  
pp. 198-209 ◽  
Author(s):  
Christopher J. Hall ◽  
Maria Vega Flores ◽  
Stefan H. Oehlers ◽  
Leslie E. Sanderson ◽  
Enid Y. Lam ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Progenitor Cell ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
Inducible Nitric Oxide

Genetic Determinants of the Definitive Hematopoietic Stem/Progenitor Cell Compartment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1226-1226
Author(s):  
Kirby D Johnson ◽  
Xin Gao ◽  
Rajendran Sanalkumar ◽  
Amy P Hsu ◽  
Myung-Jeom Ryu ◽  
...  
Keyword(s):  
Progenitor Cell ◽  
Fetal Liver ◽  
Chromatin Accessibility ◽  
Complete Loss ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
Composite Element ◽  
Fold Reduction ◽  
E Box ◽  
Definitive Hematopoiesis

Abstract Abstract 1226 How transcriptional and post-transcriptional mechanisms control the levels/activities of master developmental regulators has fundamental importance for understanding complex developmental processes such as hematopoiesis and associated pathological disorders. GATA-2 is an essential regulator of hematopoiesis, and GATA-2 mutations characterize heritable disease associated with myelodysplastic syndrome and acute myeloid leukemia, including MonoMAC (syndrome of monocytopendia, B and NK cell lymphopenia, and mycobacterial, fungal and viral infection). However, many questions remain unanswered regarding mechanisms underlying GATA-2 regulation and function. We demonstrated that a MonoMAC patient harbors a 28 bp deletion within GATA2 intron 5 that eliminates a conserved E-box and 5 base pairs of an 8 base pair spacer between the E-box and a conserved GATA motif, which constitutes an E-box-GATA composite element. This composite element resides within the +9.5 kb “GATA switch site” that binds GATA-2 and GATA-1 in the transcriptionally active and repressed states, respectively, and confers hematopoietic and vascular endothelial enhancer activities in transgenic mouse embryos. Importantly, this patient lacked mutations in the GATA2 coding sequence characteristic of other MonoMAC patients, but exhibited prototypical MonoMAC. To elucidate the mechanism underlying the function of the +9.5 composite element, we generated a targeted deletion of the murine element, which yielded embryonic lethality at E13 to E14. Prior to death, +9.5−/− mice exhibit reduced liver size, hemorrhaging, and edema. Nucleated primitive red cells are abundant in the +9.5−/− embryos, in contrast to Gata2 knockout mice, which die at approximately E10.5 from anemia due to failure of primitive and definitive hematopoiesis. Furthermore, primitive erythroid (EryP) colony assays conducted with yolk sacs revealed that the mutation does not affect primitive erythroid precursor functionality. However, the +9.5 deletion strongly reduced Gata2 expression at sites of definitive hematopoiesis, including the fetal liver (8.1 fold, P < 0.004) and cultured explants of the hematopoietic stem cell-generating Aortic Gonadal Mesonephric (AGM) region (4.0 fold, P < 0.001). The homozygous mutant animals exhibited a nearly complete loss of hematopoietic stem cells as determined by flow cytometry (20-fold reduction of Lin-Mac1+CD41-CD48-CD150+Sca+Kit+ cells, P < 0.005) and competitive repopulation (complete loss, P < 0.02) assays, as well as progenitors as determined by colony assays (BFU-E, 60-fold reduction, P < 0.002; CFU-GM, 8.8-fold reduction, P < 0.0001; CFU-GEMM, 19-fold reduction, P < 0.001). To investigate the underlying mechanisms, we developed an allele-specific Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay with heterozygous fetal liver cells to test whether the deletion influences Gata2 chromatin accessibility at the +9.5 region. The deletion significantly reduced (8.4 fold reduction, P < 0.001) chromatin accessibility at this region within the mutant allele, while the wild type allele was unaffected. Thus, any potential remaining cis-elements are insufficient to confer chromatin accessibility, supporting a model in which the transcription factors that normally occupy this GATA switch site lose the capacity to access their respective cis-elements in the context of the mutant allele. Our human and murine studies have therefore revealed a cis-element indispensable for the regulation of Gata2 expression in multiple developmental contexts and necessary for the generation of the definitive hematopoietic stem/progenitor cell compartment. As additional elements are likely to confer Gata2 expression in distinct contexts, including primitive erythropoiesis, we have implemented a multi-faceted effort to identify such elements and to compare their mechanisms with that of the +9.5 site, which will provide fundamental insights into genetic mechanisms controlling normal and malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

The Cell Cycle in Hematopoietic Stem Cells, Lin−/C-kit+/Sca1+ Fraction, Is Regulated by Connexin 32, Specifically Expressed in the Stem Cell Compartment

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4775-4775
Author(s):  
Yoko Hirabayashi ◽  
Byung-Il Yoon ◽  
Isao Tsuboi ◽  
Yan Huo ◽  
Yukio Kodama ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
In The Wild ◽  
Marrow Cells

Abstract Connexin (Cx) functions in the organization of cell-cell communication via gap junctions in multicellular organisms. Gap junctions have been implicated in the homeostatic regulation of various cellular functions, including growth control, cellular differentiation, apoptosis and the synchronization of electrotonic and metabolic functions. As Cxs are essential molecules for multicellular organisms, Cxs that organize cell-cell communication within the hematopoietic progenitor cell compartment are surmised to be present in bone marrow tissue. Recently, we first found that Cx32 is only Cx molecule expressed in the bone marrow in wild-type mice by means of comparison with Cx32-knockout (KO) mice, studied by a reverse biological approach. Cx32 is specifically expressed in primitive hematopoietic stem/progenitor cells, i.e., the lineage marker-negative (Lin−)/c-kit positive (c-kit+)/stem cell antigen-1-positive (Sca1+) (=LKS) fraction, and likely playing a role of restoration of stem/progenitor cell-quiescence, thereby preventing primitive stem cells from exhaustion. In this study, we present results on cell cycle analyses with respect to the function of Cx32; one for colony-forming progenitors by the method evaluating the cycling progenitor cells using incorporation of bromodeoxyuridine (BrdUrd) followed by ultraviolet-light cytocide and the other for primitive progenitor cells using a cell sorter with bioactive AT-rich DNA-binding dye Hoechst 33342. In the colonization assay on CFU-S-13 (primitive hematopoietic progenitor cells), the incorporation of BrdUrd starts from a higher percentage with rapid increase in Cx32-KO mice, suggesting suppression of cell cycle in these primitive hematopoietic progenitor cells with Cx32-mediated cell-cycle regulation in the wild-type steady state. This suppression may be attenuated in CFU-S-9, a differentiated progenitor cell compartment. The progenitor cells assayed by in vitro colonization on CFU-GM also showed accelerated cell cycle in the Cx32-KO mice. Following the incorporation of Hoechst 33342, the lineagedepleted bone marrow cells were analyzed by flow cytometry. The population sizes of the LKS fraction obtained were 0.052% in the Cx32-KO bone marrow cells and 0.035% in the wild-type bone marrow cells (p=0.0458&lt;0.05). The lineage-depleted bone marrow cells were analyzed their cell-cycle patterns by flow cytometry, and the G0/G1 was calculated for the LKS fractions in both, the Cx32-KO mice and wild-type mice. The percentage of G0/G1 calculated for the LKS fractions were significantly lower in the Cx32-KO mice than those in wild-type mice (60.6% vs. 87.9% for Cx32-KO vs. wild-type; p=0.001). The results suggest that Cx32 may have suppressive functions on the hematopoietic stem cell compartment, the LKS fraction, under the physiological function of Cx32. The Cx32 in the wild-type mice is, thus considered to be expressed in the primitive hematopoietic stem/progenitor cells to prevent from their exhaustion.

Modelling Cellular and Molecular Progression Of CML In The Mouse

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2706-2706
Author(s):  
George Giotopoulos ◽  
Louise Van Der Weyden ◽  
Hikari Osaki ◽  
Wai-In Chan ◽  
Alistair Rust ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Disease Progression ◽  
Myeloid Leukemia ◽  
Progenitor Cell ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Mutation Induction ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, caused by a reciprocal chromosomal translocation that generates the BCR-ABL fusion protein, a constitutively activated tyrosine kinase. Patients with CML usually present in an indolent chronic phase (CP), however, if left untreated, they irrevocably progress to an aggressive form of acute leukemia, termed blast crisis (BC) that is usually fatal. Tyrosine kinase inhibitor (TKI) (e.g. Imatinib) treatment has revolutionised the treatment of CML CP. However, ∼5-10% of CP patients will progress to BC despite TKI treatment, and an additional 10-15% of patients are beyond CP at initial presentation. Upon disease progression, treatment options are very limited and prognosis is dismal. Hence, understanding the events that drive disease progression and identifying potential therapeutic targets remains an unmet clinical need. The mechanisms of BC transformation are poorly understood, but it is generally accepted that additional somatic mutations are required. To date, a small number of recurrent mutations have been reported, but these only account for a relatively small number of cases and their exact nature is not fully understood. In order to study the mechanisms of BC progression and to identify the co-operating mutations that drive this, we have utilised a published transgenic murine model of chronic phase CML (Koschmieder et al., 2005) and performed a transposon-based forward insertional mutagenesis study. In our mouse model, expression of BCR-ABL was driven in the hematopoietic stem and progenitor cell compartment (HSPC) by an SCL enhancer in a tetracycline dependant manner. Following BCR-ABL expression we conditionally induced ongoing mutations via a transposon-transposase system (SB) within HSPC and monitored disease progression from the chronic/BCR-ABL dependant phase to the transposon-mediated BC. Utilising the design of the transposon based system, it was then possible to identify these mutations by multiplexed next generation sequencing (NGS). Our experimental cohort was comprised of BC mice (which expressed BCR-ABL and transposon/transposase mediated mutation induction), CML mice (BCR-ABL only) and SB mice (mutation induction only). BC mice demonstrated a significantly shorter survival (p<0.0001, 116 vs. 147 days) compared to CML mice. Disease progression was characterised by a significantly increased disease burden, in terms of organ infiltration and leucocytosis, with around 80% of BC mice developing an exclusively acute myeloid leukemia by the Bethesda criteria. BC mice also demonstrated quantitative and functional differences within the hematopoietic stem and progenitor cell compartment in in vitro and in vivo assays in keeping with progression from a chronic to an acute leukemia. Importantly, BC mice showed a shorter survival (p=0.007, 116 vs. 128 days) compared to the SB mice, in which both acute myeloid and lymphoid leukemias were seen. Molecularly, NGS revealed insertions in both novel genes, and in genes previously implicated in CML blast crisis, hematopoiesis and leukemogenesis, such as ASXL1, FLT3 and ERG. These insertions included highly recurrent hits and were enriched for transcriptional regulators and signalling proteins, many of potential therapeutic relevance. Additionally, there was only a very modest overlap between the mutations identified in the BC and the SB cohorts, demonstrating BCR-ABL-dependant cooperation and disease progression. Considering the above data, our mouse model shows great potential in understanding the mechanisms of transformation to blast crisis, and ultimately in identifying potential therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

289: Roles of Inducible Nitric Oxide Synthase in Voiding Function and Bladder Pain During Experimental Cystitis

2006 ◽  
Vol 175 (4S) ◽  
pp. 96-96
Author(s):  
Masayoshi Nomura ◽  
Hisae Nishii ◽  
Masato Tsutsui ◽  
Naohiro Fujimoto ◽  
Tetsuro Matsumoto
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Voiding Function ◽  
Bladder Pain ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide
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