Hematopoiesis In Mouse Embryo Research Articles

7260 Nuclear Corepressor 1 (NCoR1) plays important role in the regulation of insulin gene transcription in mouse Min6 pancreatic beta cells

Abstract Disclosure: H. Shimizu: None. Y. Horibata: None. I. Amano: None. M.J. Ritter: None. M. Ohyama: None. M. Domae: None. H. Sugimoto: None. A.N. Hollenberg: None. Background: Nuclear Corepressor 1 (NCoR1) is a transcriptional corepressor which has been recognized as an important player in the regulation of hepatic lipogenesis and hematopoiesis in mouse embryo. NCoR1 is also widely expressed in mouse insulin sensitive organs, for instance adipocyte, skeletal muscle, and hepatocytes. Mouse genetic deletion strategies of either NCoR1 or SMRT(NCoR2; an ortholog corepressor of NCoR1) demonstrated that each corepressor effects the insulin sensitivity in the tissues. However, the role of each corepressor in pancreatic beta cell is not still elucidated. Methods: To clarify this, we took the gene-targeting strategy for NCoR1 using CRISPR Cas-9 and made the pancreatic beta Min6 clone which lacks NCoR1 protein (Min6-NCoR1KO; NKO cells). For this study, wild type Min6 cells (WT) and NKO cells were cultured in the Dulbecco's DMEM medium with 10% fetal bovine serum for 4-5 days, and the expression analyses compared between two cell types were performed. Results: Both comprehensive proteomics and following expression analyses compared between WT and NKO cells demonstrated that NKO cells have significantly lower expression level of insulin (INS-1 and INS-2), accompanied with high expression of histone deacetylase 6 (HDAC6). For further analyses, we also made the Min6 clone which lacks SMRT(Min6-SMRTKO; SKO cells). Interestingly, SKO cell had higher insulin and lower HDAC6 levels, accompanied with significant up-regulation of NCoR1. Conclusion: Taken together, these data indicate that NCoR1 has dominant effects in the regulation of both histone deacetylation and insulin gene transcription in Min6 cells. Further investigations for the function of NCoR1 in the insulin gene transcription observed in Min6 cells will be needed. Presentation: 6/2/2024

Loss of DHX15 Function Impairs Hematopoiesis in Vav-Dhx15-KO Mice and Cause DNA Damage-Induced Cell Cycle Arrest and Apoptosis in Leukemia Cell Lines

DHX15, a member of the DEAD-box RNA helicase family, acts as splicing factor in pre-messenger RNA splicing, pre-ribosomal RNA, and RNA metabolism. Our previous studies showed that DHX15 overexpression is associated with poor prognosis in acute myeloid leukemia (AML), and DHX15 regulates cell apoptosis via the NF-kB signaling pathway(Pan et al., 2017). However, its molecular function remains obscure in the blood system. First, with the Cre-loxp knockout system, Dhx15fl/fl male mice were crossed with vav-icre female mice (specifically expressed in blood system), we found that Dhx15fl/fl; vav-icre homozygous knockout mice (vav-Dhx15-KO) are lethal by E17.5-E18.5. Then, to explore the inner difference in blood system, the hematopoietic stem cells (HSC) were analyzed by flow cytometry in fetal liver (FL) (E13.5, E15.5). Compared with Dhx15fl/fl (wild-type, WT) mice, the lineage-negative cells (Lin- cells; Lin+ Cocktail, CD5, CD45R (B220), CD11b, Anti-Gr-1 (Ly-6G/C), 7-4 and Ter-119) significantly increased in vav-Dhx15-KO FL cells (P<0.0001) and the short-term HSC(ST-HSC) proportion compensatively increased in LSK (Lin-c-Kit+Sca1+cells) population (P<0.01), while the proportion of LK(Lin-c-Kit+Sca1-cells) (P<0.0001 in E13.5, P<0.001 in E15.5), LSK(Lin-c-Kit+Sca1+cells)(P<0.1 in E15.5), and multipotent progenitor (MPP) (P<0.0001 in E15.5) all witnessed decrease in the Lin-/LSK population respectively. Meanwhile, vav-Dhx15-KO mice exhibited impaired multi-lineage hematopoiesis especially in erythroid, where the precursors and immature erythrocytes increased, mature erythrocytes decreased (P<0.001 in E13.5). Next, in vivo competitive repopulation assay were performed to further examine any potential cell-intrinsic defect of HSC function caused by Dhx15-inactivation. Thus, 1,000,000 E13.5/E15.5 total FL donor cells (CD45.2, vav-Dhx15-KO or WT) together with 1,000,000 CD45.1 unfractionated BM competitor cells were intra-venous transplanted into lethally irradiated (9Gy) B6.SJL (CD45.1) recipient mice. Recipient mice were sacrificed 8-weeks after transplantation, flow cytometry was conducted to analysis the ratio of CD45.2 in mice, the results showed that vav-Dhx15-KO FL was severely deficient compared to WT FL(0.1±0.04%vs 62.2±2.8% in BM, 0.2±0.05% vs 64.5±6.1% in SP, 0.1±0.05% vs 45.1±5.5% in PB). These results indicate that Dhx15 is crucial in mice blood system, loss of its function may block cell differentiation, leading to the reduction of mature cells. At the same time, to further explore the molecular mechanism of DHX15 in blood system, MOLM-13 and NB4 leukemia cell lines were selected for shRNA-KD (knockdown) experiments due to the lethality of vav-Dhx15-KO mice. We found that Dhx15-KD significantly impaired the proliferation ability of these cell lines in vitro and in vivo, by experiments of survival competition, clone formation, subcutaneous and intra-venous tumorigenesis. Besides, our data demonstrate DHX15-KD-mediated G0/G1 cell cycle arrest and increase in early-late apoptotic cells by Hoechest 33342-EdU cell cycle and DAPI-Annexin V cell apoptosis assays. Mechanistically, we found that UV_RESPONSE signaling pathway which is mainly related to DNA damage was up-regulated by RNAseq and bioinformatics analysis (DHX15-KD cell line vs WT cell line). Correspondingly, the DNA damage indicators γH2AX and 53BP1 were also increased in DHX15-KD cell lines detected by WB and immunofluorescence experiments, suggesting that the deletion of DHX15 could lead to DNA damage-mediated cell cycle arrest and apoptosis in leukemia cell lines. In summary, these results show that DHX15 deficiency leads to DNA damage-induced cell cycle arrest and apoptosis which further impairs the proliferative capacity of leukemia cell lines and lost function of Dhx15 impairs multi-lineage hematopoiesis in mouse embryos, which may lead to its perinatal death.

LRF-mediated Dll4 repression in erythroblasts is necessary for hematopoietic stem cell maintenance

AbstractHematopoietic stem cells (HSCs) are the most primitive cells in the hematopoietic system and are under tight regulation for self-renewal and differentiation. Notch signals are essential for the emergence of definitive hematopoiesis in mouse embryos and are critical regulators of lymphoid lineage fate determination. However, it remains unclear how Notch regulates the balance between HSC self-renewal and differentiation in the adult bone marrow (BM). Here we report a novel mechanism that prevents HSCs from undergoing premature lymphoid differentiation in BM. Using a series of in vivo mouse models and functional HSC assays, we show that leukemia/lymphoma related factor (LRF) is necessary for HSC maintenance by functioning as an erythroid-specific repressor of Delta-like 4 (Dll4) expression. Lrf deletion in erythroblasts promoted up-regulation of Dll4 in erythroblasts, sensitizing HSCs to T-cell instructive signals in the BM. Our study reveals novel cross-talk between HSCs and erythroblasts, and sheds a new light on the regulatory mechanisms regulating the balance between HSC self-renewal and differentiation.

Fibroblastic Potential of CD41+Cells in the Mouse Aorta-Gonad-Mesonephros Region and Yolk Sac

CD41 (αIIb integrin), a specific marker for megakaryocytes and platelets, was recently shown to mark the initiation of definitive hematopoiesis in mouse embryos. However, whether embryonic CD41(+) populations have a nonhematopoietic potential remains elusive. Here, we report that the CD41(+) cells from the mouse E11.0 aorta-gonad-mesonephros (AGM) region and yolk sac (YS) expressed a set of mesenchymal markers (as revealed by reverse transcriptase-polymerase chain reaction), displayed myofibroblastic/fibroblastic potential in vitro under mesenchymal culture conditions, and differentiated into α-SMA(+)/epimorphin(+)/vimentin(+) cells in the lungs of adult recipients after systemic transplantation. This unique cell population with fibroblastic potential expressed intermediate rather than high levels of CD41 and was negative for CD34 in the AGM region. In contrast, circulating CD41(+) cells in the embryonic blood stream harbored no similar fibroblastic potential. Compared with the YS, the AGM-derived CD41(+) cells had a more robust fibroblastic potential, as revealed by higher in vitro growth rates. Interestingly, the AGM-derived CD41(+) cells demonstrated a stronger response to the chemotaxin of circulating blood plasma than the YS-derived CD41(+) cells. We are the first group that illustrates the fibroblastic potential of an embryonic CD41(+) population in vitro and in vivo, reflecting the close association between blood and mesenchyme development during mouse mid-gestation. The precise origin of these mesenchymal populations needs further clarification.

AML1/Runx1 Negatively Regulates Quiescent Hematopoietic Stem Cells in Adult Hematopoiesis

Transcription factor AML1/Runx1, initially isolated from the t(8;21) chromosomal translocation in human leukemia, is essential for the development of multilineage hematopoiesis in mouse embryos. AML1 negatively regulates the number of immature hematopoietic cells in adult hematopoiesis, whereas it is required for megakaryocytic maturation and lymphocytic development. However, it remains yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population fraction are increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We also found an increase in HSC number within the AML1-deficient bone marrow using limiting dilution bone marrow transplantation assays. These results indicate that the number of quiescent HSCs is negatively regulated by AML1.

Implication of AML1/RUNX1 Function in the Homeostasis and Leukemic Transformation of Hematopoietic Stem Cells.

Transcription factor AML1/RUNX1, initially isolated from the t(8;21) chromosomal translocation in human leukemia, is essential for the development of multilineage hematopoiesis in mouse embryos. AML1 negatively regulates the number of immature hematopoietic cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. However, it remains yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population fraction and c-Kit-positive cells in the G0 cell cycle status were increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We also found an increase in HSC number within the AML1-deficient bone marrow using limiting dilution bone marrow transplantation assays. Thus, the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia. To identify mechanisms through which functional loss of AML1 exerts leukemogenic potential, we focused on the AML1-Evi-1 chimeric protein, which is generated by the t(3;21) chromosomal translocation and disturbs the normal function of AML1. We introduced AML1-Evi-1 and its mutants into murine bone marrow cells, and evaluated hematopoietic cell transformation by colony replating assays. The transforming activity of AML1-Evi-1 was impaired when any of the major functional domains of AML1-Evi-1 was lost. Moreover, overexpression of Evi-1 could not transform AML1-deleted bone marrow cells, suggesting that fusion of AML1 and Evi-1, rather than AML1 suppression and Evi-1 overexpression, is essential for AML1-Evi-1 leukemogenesis. Intriguingly, among the hematopoietic progenitor cell fractions, AML1-Evi-1 could transform only the uncommitted, immature hematopoietic cells, which contrasts with MLL-ENL, a chimeric protein generated in t(11;19) leukemia. AML1-Evi-1 transformed cells show a surface marker profile different from that of the cells transformed by AML1-MTG8/ETO, another leukemic gene product that also perturbs AML1 function. These results provide a valuable clue to a distinct mechanism determined by the Evi-1 moiety in the AML1-Evi-1 leukemogenesis and to a role of AML1 loss in the self-renewal of leukemic stem cells.

AML1/Runx1 Negatively Regulates the Number of Quiescent Hematopoietic Stem Cells in Adult Hematopoiesis.

Transcription factor AML1 (also called Runx1), which was initially isolated from the t(8;21) chromosomal translocation frequently found in the acute myelogenous leukemia FAB M2 subtype, is essential for the development of multilineage hematopoiesis in mouse embryos. By analyzing conditional AML1 knockout mice, we have previously shown that AML1 negatively regulates the number of immature hematopoietic cells defined as lineage-negative, CD34− Sca-1+ c-Kit+ (34KSL) cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. The former is a significant observation because an increase in hematopoietic stem/progenitor cells due to defective AML1 function may be closely related to the development of human leukemia. In support of this is the fact that mice in which leukemic chimeric protein AML1/ETO is expressed in hematopoietic cells are subject to myeloproliferative disease and develop leukemia after additional mutation. However, it has remained yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population (SP) fraction are increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We quantitatively evaluated HSCs by bone marrow transplantation assays using limiting dilution and found a significant increase in HSC number within the AML1-deficient bone marrow. These results indicate that the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia.

Inhibitory effects of homeodomain-interacting protein kinase 2 on the aorta–gonad–mesonephros hematopoiesis

Definitive hematopoiesis starts in the aorta–gonad–mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45 + hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45 lowc-Kit + cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo.

Expression of Runx1 Isoforms in Sub-Populations of Human Blood and Bone Marrow Cells.

Runx1 is a pivotal regulator of hematopoiesis. Deficiency of Runx1 leads to complete block of definitive hematopoiesis in mouse embryos. Runx1 protein forms a heterodimer with CBFβ and binds to the specific DNA sequence TGT/cGGT to regulate the expression of a number of hematopoietic genes. Runx 1 has been recognized to have three different isoforms (a, b, and c) due to differential splicing and promoter utilization. The distal (Runx1c) promoter region was shown to have binding sites mediating repression of Runx1 transcription. Data on the pattern of Runx1 isoforms expression in different hematopoietic lineages are scant. The functional impact of different Runx1 isoforms on commitment and differentiation of blood cells is also essentially unknown. To further explore this, we studied Runx1 isoform expression in different subpopulations of human hematopoietic cells. Total RNA was extracted from peripheral blood and unstimulated bone marrow. Also, Lin-CD34+CD38+, and Lin-CD34+CD38− cell populations were sorted from G-CSF-stimulated bone marrow and total RNA extracted. Expression of Runx1 isoforms was assessed by semiquantitative RT-PCR analysis, using GAPDH as a control. All 3 isoforms were present in all studied cell populations. Runx1a was uniformly a minor form with lowest expression. Runx1c isoform was a dominant one in peripheral blood as opposed to Runx1b in bone marrow. Most interestingly, the ratio of Runx1b to other isoforms was significantly increased in Lin-CD34+CD38- subpopulation compared to Lin-CD34+CD38+ cells. The prevalence of Runx1c isoform in peripheral blood as compaired to bone marrow is most likely related to a different cellular composition of these tissues and suggests that Runx 1c isoform may be predominantly expressed in some of the mature cell lineages. Since Lin-CD34+CD38− cells represent most primitive progenitor cell population with multi-lineage repopulating ability, the observed overexpression of Runx1b may be related to transcriptional accessibility of these cells. Upregulation of Runx 1b with G-CSF in this subpopulation can not be ruled out as well. These preliminary data suggest that the expression of Runx1 alternative splicing isoforms is both lineage and differentiation stage-specific in human hematopoietic cells. We will also test whether Runx1 isoform expression has a consistent pattern in different types of leukemia and if it is resembles such of mature blood counterparts or primitive progenitors. These studies, as well as the studies of differential expression of Runx1 isoforms in different mature blood lineages and progenitor cell sub-populations are underway and results will be presented. These data may further shed a light on functional differences in Runx1 isoforms and mechanisms of hematopoiesis.

Fetal Hematopoietic Development in the Mouse Aorta-Gonad-Mesonephros Region Is Inhibited by Homeodomain-Interacting Protein Kinase 2.

Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of mouse embryo. Our previous studies showed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine 727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in the hematopoietic development. HIPK2 transcripts were found in the fetal hematopoietic tissues such as the AGM region at embryonic day (E) 10.5 and E11.5 and the E14.5 fetal liver as detected by RT-PCR and whole mount in situ hybridization. The functional analysis of HIPK2 was done by introducing wild-type and its mutant constructs into the primary culture of the mouse AGM region. Retrovirus mediated forced expression of HIPK2 with green fluorescent protein (GFP) in the AGM culture remarkably inhibited the emergence of GFP+ nonadherent cells. In coculture of virus-infected GFP+ nonadherent cells with OP9 stromal cells, no cobblestone-like area-forming colonies were detected in the HIPK2-infected cells, while control GFP virus had no effect on the colony formation. Similarly, when GFP+ nonadherent cells were sorted and cultured in semisolid medium, colonies were not formed from the HIPK2-GFP virus-infected AGM culture. These data suggest that HIPK2 suppresses differentiation of adherent endothelial-like cells to colony-forming hematopoietic cells or, alternatively HIPK2 inhibits proliferation of newly developed hematopoietic progenitors. Development of cells positive for CD45 (an immature hematopoietic cell maker) cells was suppressed by forced expression of HIPK2 in the AGM culture. To determine the domain important for the inhibitory activity of HIPK2, we constructed a series of point and deletion mutants. Overexpression of HIPK2 mutants in the AGM culture showed that a certain structure of the kinase domain as well as its C-terminally located structure but not the kinase activity is essential for the HIPK2-mediated inhibition of hematopoiesis. When nonadherent cells in the AGM culture were fractionated for their expression levels of CD45 and c-Kit (a receptor for stem cell factor), CD45lowc-Kit+ cells have a potential to form hematopoietic cell colonies. In the HIPK2-overexpressed AGM culture, emergence of CD45lowc-Kit+ cells was significantly inhibited. These data indicated that HIPK2 plays a negative regulatory role in the development of AGM hematopoiesis in mouse embryo.

Cbfβ is involved in maturation of all lineages of hematopoietic cells during embryogenesis except erythroid

The transcription factor Cbfβ forms a heterodimeric complex with members of the Runx family of proteins. Together, Cbfβ and Runx1 play a critical role in the establishment of definitive hematopoiesis in mouse embryos. Previously, we used a Cbfb-GFP “knock-in” mouse model to demonstrate that Cbfβ is expressed in hematopoietic stem cells of the mouse fetal liver and aorta-gonad-mesonephros (Blood 100 (2002), 2449). We also examined the expression pattern of Cbfβ in different lineages of adult hematopoietic cells and found that it is expressed uniformly in all lineages except B lymphocytes and erythroid cells. Cbfβ expression decreases during maturation of B cells in the adult bone marrow, and is not expressed in nucleated erythroid precursors. Here, we examine the expression of Cbfβ in various hematopoietic lineages, including myeloid, lymphoid, and erythroid during late stages of embryonic development, and compare it to the pattern observed in adults. We find that there are subtle differences in expression of Cbfβ-GFP in embryonic hematopoietic cells compared to their adult counterparts, but that the overall pattern is the same. Our data complement recently published data on hematopoetic defects observed in transgenic Cbfb-null mouse embryos partially rescued by ectopic expression of Cbfb (Nature Genet. 32 (2002), 633; Nature Genet. 32 (2002), 645). and supports the emerging view that Cbfβ and Runx proteins are required for normal maturation of hematopoietic cells as well as establishment of definitive hematopoiesis.