Mouse Fetal Liver Research Articles

Reprogramming to iPS cells and their subsequent hematopoietic differentiation is more efficient from MEFs than from preB cells

Efficiencies of the generation of induced pluripotent stem (iPS) cells from either mouse embryonic fibroblasts (MEF) or from mouse fetal liver (FL) derived preB cells and their hematogenic potencies were compared. In 10 days approximately 2% of the MEFs transduced with Sox-2, Oct-4 and Klf-4 developed to iPS cells, while only 0.01% of transduced FL-preB cells yielded iPS cells, and only after around 3 weeks. Subsequently, the generated iPS cells were induced to differentiate into hematopoietic cells in vitro. On day 5 of differentiation MEF-iPS yielded numbers and percentages of Flk-1+ mesodermal-like cells comparable to those developed from embryonic stem (ES) cells. Compared to ES cells further differentiation to hematopoietic and lymphopoietic cells was reduced, possibly because of persistent expression of the reprogramming factors. By contrast, FL-iPS cells developed lower numbers and percentages of Flk-1+ cells, and no significant further development to hematopoietic or lymphopoietic cells could be induced. These results indicate that the efficiencies of iPS generation and subsequent hematopoietic development depends on the type of differentiated cell from which iPS cells are generated.

Increased Reprogramming Capacity of Mouse Liver Progenitor Cells, Compared With Differentiated Liver Cells, Requires the BAF Complex

Ectopic expression of certain transcription factors can reprogram somatic cells to a pluripotent state. Hematopoietic and muscle stem cells can be more efficiently reprogrammed than differentiated blood or muscle cells, yet similar findings have not been shown in other primary organ systems. Moreover, molecular characteristics of the cellular hierarchy of tissues that influence reprogramming capacities need to be delineated. We analyzed the effect of differentiation stage of freshly isolated, mouse liver cells on the reprogramming efficiency. Liver progenitor cell (LPC)-enriched cell fractions were isolated from adult (6-8 wk) and fetal (embryonic day 14.5) livers of mice and reprogrammed to become induced pluripotent stem (iPS) cells. Different transcription factors were expressed in liver cells, and markers of pluripotency were examined, along with the ability of iPS cells to differentiate, in vitro and in vivo, into different germ layers. Fetal and adult LPCs had significantly greater reprogramming efficiency after transduction with 3 or 4 reprogramming factors. Transduction efficiency-corrected reprogramming rates of fetal LPCs were 275-fold higher, compared with unsorted fetal liver cells, when 3 reprogramming factors were transduced. The increased reprogramming efficiency of LPCs, compared with differentiated liver cells, occurred independently of proliferation rates, but was associated with endogenous expression of reprogramming factors (Klf4 and c-Myc) and BAF (Brg1/Brm associated factor)-complex members Baf155 and Brg1, which mediate epigenetic changes during reprogramming. Knockdown of BAF complex members negated the increased reprogramming efficiency of LPCs, compared with non-LPCs. LPCs have intrinsic, cell proliferation-independent characteristics resulting in an increased reprogramming capacity compared to differentiated liver cells.

Distinct In Vitro Myelopoiesis is Dependent on the Self‐Renewal of Hematopoietic Progenitors

Spleen and bone marrow (BM) have been shown to contain the progenitors of a novel dendritic-like antigen-presenting cell type (L-DC). These progenitors are also maintained in both long-term spleen cultures and co-cultures of spleen or BM over the stromal cell line STX3. We examined mouse foetal liver (FL), rich in hematopoietic stem/progenitor cells (HSC/HPC) after embryonic day (E) 12.5, for the presence of L-DC progenitors by testing their capacity to colonize STX3 and produce L-DC. E14.5 FL from wild-type C57BL/6J mice was found to colonize STX3 and produce L-DC for 28 days. By contrast, E14.5 FL from Ikaros Plastic mice gave only short-term production of low numbers of L-DC between 7 and 14 days of co-culture. The transient and weak production of L-DC by FL from Plastic E14.5 mice maps to the loss of self-renewal capacity amongst HSC. L-DC progenitors are, therefore, closely aligned with a subset of self-renewing HSC/HPC in FL.

Mutant HrasG12V and KrasG12D have overlapping, but non‐identical effects on hepatocyte growth and transformation frequency in transgenic mice

Mouse hepatocarcinogenesis is associated with mutations in Hras, but infrequently in Kras. The effect on carcinogenesis of developmental age at the time of ras mutation remains unknown. We sought to compare quantitatively the effects of expressing mutant H- or Kras genes in fetal vs. adult mouse liver. We established an inducible system of gene expression in mouse liver to define disease pathogenesis associated with activation of oncogene expression. Diffuse expression of either oncogene in fetal or adult hepatocytes caused hepatomegaly. For mutant Hras(G12V), this phenotype was almost fully reversible and accompanied by apoptosis, indicating that maintenance of hepatomegaly requires continuous Hras(G12V) expression. We also examined the effect of ras expression on growth of transplanted hepatocytes in an in vivo system that allows us to quantify hepatocyte growth effects in both permissive and restrictive hepatic growth environments. Mutant Kras(G12D) had no effect on hepatocyte growth in this system. In contrast, Hras(G12V) induced increased hepatocyte focus growth in quiescent liver, the hallmark of a cell autonomous growth stimulus. Hras(G12V) also increased the fraction of donor hepatocyte foci that displayed extreme growth, a characteristic of preneoplastic lesions. The primary effect of diffuse, whole-liver expression of either mutant ras gene in fetal or adult mouse liver is diffuse and progressive hepatic growth. Hras(G12V) mutation influences hepatocarcinogenesis by conferring cell autonomous growth potential upon foci of expressing cells and by increasing the risk of neoplastic progression. Kras(G12D) does not share these latter carcinogenic effects in mouse liver.

Transplantation of mouse fetal liver cells for analyzing the function of hematopoietic stem and progenitor cells.

Hematopoietic stem cells are defined by their ability to self-renew and differentiate through progenitor cell stages into all types of mature blood cells. Gene-targeting studies in mice have demonstrated that many genes are essential for the generation and function of hematopoietic stem and progenitor cells. For definitively analyzing the function of these cells, transplantation studies have to be performed. In this chapter, we describe methods to isolate and transplant fetal liver cells as well as how to analyze donor cell reconstitution. This protocol is tailored toward mouse models where embryonic lethality precludes analysis of adult hematopoiesis or where it is suspected that the function of fetal liver hematopoietic stem and progenitor cells is compromised.

Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Abstract 344Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free’ heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias. Disclosures:No relevant conflicts of interest to declare.

The TLE1 Tumor Suppressor Regulates Myc Induced Leukemogenesis

Abstract 2473The Groucho/TLE family of corepressors has been described as master regulatory genes during development, affecting multiple cell fate decisions. These proteins bind to a variety of transcription factors and recruit inhibitory proteins to repress transcription. We previously identified TLE1 as a novel tumor suppressor gene that is deleted or methylated in subgroups of acute myeloid leukemia (AML) and other hematological malignancies. We find the loss of Tle1 alone is insufficient to induce leukemia in mice and apparently requires cooperation with additional oncogenes. Our studies, and those from other groups, have shown that over-expression of TLE1 in leukemia cells slows cell cycle progression, colony formation and tumor growth in xenografts, while silencing results in increased cell proliferation. The pathways by which TLE1 affects oncogenesis is unclear, but this gene family is capable of interacting with effectors of Myc, Wnt, Notch, TGFB signaling–prominent pathways dysregulated in malignancies.Myc is important for hematopoietic stem cell proliferation, survival and differentiation and is over-expressed in most AML samples. The TLE homologue Groucho binds and represses Drosophila Myc expression of target genes, thus we postulated that TLE1 could be an important regulator of Myc activity in leukemia. Using hematopoietic progenitor cells from Tle1 knockout and wild-type fetal livers we found that the loss of Tle1 dramatically increased proliferation and serial replating efficiency. Expression of N-Myc by itself in wild type fetal liver cells triggered significant cell death and apoptosis. However, when N-Myc expression was combined with the additional loss of Tle1, not only was N-Myc induced apoptosis inhibited, but a dramatic cell proliferation, well in excess of that seen with Tle1 loss by itself, was seen. Furthermore, mice transplanted with N-Myc transduced hematopoietic cells from Tle1 knockout mice fetal liver developed a more aggressive leukemia, compared to N-Myc transfected wild type mice fetal liver hematopoietic cells, with increased proliferation of leukemic cells as demonstrated by in vitro colony assays and higher secondary transplantability.We extended these studies to a zebrafish model of Rag2-Myc mediated T-ALL. Using these zebrafish we demonstrated over-expression of the TLE homologue, Groucho, completely blocked the initiation and progression of Myc induced leukemia development. Expression of a truncated version of Groucho reduced the initiation of T-ALL and prolonged the survival of fish developing leukemia. These studies demonstrate TLE1 can inhibit the oncogenicity of Myc, and suggests modulation of expression of this gene family may be of importance for a variety of malignancies. Disclosures:No relevant conflicts of interest to declare.

Correction of Murine Sickle Cell Disease Through Interference with Fetal Hemoglobin Silencing

Abstract 351The switch from fetal (HbF, α2γ2) to adult (HbA, α2β2) hemoglobin is critical to the pathogenesis of the major hemoglobin disorders, sickle cell disease (SCD) and β-thalassemias. Increased HbF expression ameliorates the clinical severity of these conditions, forming the basis for the goal of elucidating mechanisms of globin switching and developing target-based therapies to enhance HbF production. The rationale for this therapeutic approach rests on the hypothesis that manipulation of a single target would lead to sufficient HbF reactivation for clinical benefit. Despite recent advances in understanding the complex processes of developmental globin switching and HbF silencing, this hypothesis is untested. The transcription factor BCL11A is a critical mediator of HbF switching and silencing. Previously we showed that BCL11A controls globin switching in mice and silencing of HbF expressed from human β-globin locus transgenes in mouse fetal liver, and contributes to HbF silencing in primary human erythroid cells. Furthermore, we demonstrated that BCL11A is required in vivo for HbF silencing in adult animals, yet dispensable for red cell production. These findings illustrate favorable features of BCL11A as a potential target for reactivation of HbF in the β-hemoglobinopathies.Here, we test whether impairment of BCL11A is sufficient to ameliorate disease symptoms in a relevant preclinical model of a major β-hemoglobin disorder, the humanized SCD mouse model (“Berkeley” SCD model, Paszty et al. Science 278:876–878, 1997). These mice express exclusively human sickle hemoglobin and faithfully recapitulate the principal hematologic and histopathologic phenotypes of patients with SCD. We introduced BCL11A-null alleles into the “Berkeley” SCD model (SCD Bcl11afl/fl EpoR-Cre+, hereafter called SCD/Bcl11a−/− mice). Loss of BCL11A in SCD animals restored the hematologic deficits to normal values. Compared with sickle animals, SCD/Bcl11a−/− mice have marked increases in RBC counts (6.4 ± 0.5 to 9.8 ± 0.4 × 1012/L; non-sickle control 10.1 ± 0.2 × 1012/L ) and hemoglobin level (7.8 ± 0.6 to 13.6 ± 0.7 g/L; non-sickle control 13.1 ± 0.3 g/L), accompanied by a significant reduction in reticulocyte counts (38.2% ± 3.9% to 7.0% ± 0.3%; non-sickle control 3.1% ± 0.6%). Consistent with correction of hematologic parameters, sickled erythrocytes were absent in the peripheral blood of SCD/Bcl11a−/− mice and red cell lifespan was substantially normalized. In addition, tissue damage in multiple organs was prevented in SCD/Bcl11a−/− mice. Urine osmolality of SCD/Bcl11a−/− mice was restored (from 1037 ± 82 mOsm in SCD mice to 2133 ± 333 mOsm in SCD/Bcl11a−/− mice), indicating improved renal function.To assess the extent to which these phenotypic findings were related to HbF induction, we determined the level of γ-globin expression and cellular distribution of HbF in SCD/Bcl11a−/− mice. Compared with SCD mice, expression of γ-globin genes was markedly elevated in adult SCD/Bcl11a−/− mice (1.3% to 28.3% of total β-like human globins; P = 0.00017). Peripheral blood of control and SCD mice contained few F cells (3.6% and 7.2%, respectively). In contrast, peripheral blood of SCD/Bcl11a−/− mice displayed strong pancellular staining of HbF, and F cells contributed to 85.1% of total RBCs (P = 3.9 × 10−5). The level of HbF expression achieved in the absence of BCL11A is well beyond that needed to impede HbS polymerization.In conclusion, we demonstrate that inactivation of a single component involved in HbF regulation, the transcription factor BCL11A, provides phenotypic correction of SCD in a mouse model. Our findings provide a crucial “proof of principle” for targeted reactivation of HbF as a realistic therapeutic goal. Disclosures:No relevant conflicts of interest to declare.

Deregulated Activity of AML1/RUNX1 Cooperates with BCR-ABL to Immortalize Hematopoietic Progenitor Cells and Induces Blast Crisis-Like Disease of Chronic Myelogenous Leukemia in Mice,

Abstract 3749Since the introduction of imatinib treatment for chronic myeloid leukemia (CML), most patients in the chronic phase (CP) achieve long-lasting hematologic remission. However, some patients acquire resistance or intolerance to imatinib, which results in disease progression to blast crisis (BC). Although it is known that BCR-ABL induces genomic instability, which results in leukemia cells creating multiple gene defects, genes inducing the progression to BC in CML are largely unknown. Moreover, mutations in the AML1/RUNX1 gene and overrepresentation of AML1/RUNX1 in BC compared to CP have been revealed. We therefore hypothesized that deregulation of AML1/RUNX1 might be one of the mechanisms for disease progression of CML.We initially sought to establish a culture system in vitro that allows rapid identification of genes possibly contributing to the emergence of BC. Given that cells leading to the origin of BC are known to resemble committed progenitor rather than stem cells, we employed Sca-1(-)Lin(-) progenitors derived from mouse fetal liver for the culture. Thus, cells were retrovirally transduced with BCR-ABL alone or in combination with a gene of interest, and cultured in vitro without cytokines. BCR-ABL alone-transduced cells grew initially, but ceased to do so after a week. In contrast, cells co-transduced with BCR-ABL and either one of NUP98-HOXA9, Hes1 or Bmi1 (genes known to induce BC-like disease in concert with BCR-ABL) kept growing and were immortalized in the cytokine-free culture. Importantly, cells cultivated in vitro caused fetal disease when transplanted into mice. These findings suggest the utility of our in vitro culture system as an assay of the co-operativity of a given gene with BCR-ABL to induce BC.We therefore applied the culture system for the assay of AML1/RUNX1 mutants. We chose four AML1/RUNX1 mutants (K83Q, R139G, R80C, and D171N) for the analysis of their co-operativity with BCR-ABL. Wild-type AML1/RUNX1 and a vector-only control were also included. Results showed that K83Q and R139G mutants co-operated with BCR-ABL to immortalize progenitor cells in cytokine-free culture in vitro. In contrast, R80C and D171N mutants lacked such ability. The cells co-expressing BCR-ABL and either K83Q or R139G mutants cultivated in vitro caused fetal disease in mice following transplantation: all 5 mice transplanted with BCR-ABL/K83Q-transduced cells and 7 of 10 mice transplanted with BCR-ABL/R139G-transduced cells developed lethal hematologic diseases 7–79 days following transplantation. These findings are suggestive of variance among mutants of AML1/RUNX1 regarding the ability to induce BC.Unexpectedly, further analyses revealed that wild-type AML1/RUNX1 was as potent as K83Q and R139G in inducing BC: all 4 mice transplanted with BCR-ABL/wild-type AML1-transduced cells developed fetal disease. Examination of various lengths of C-terminally truncated versions of AML1/RUNX1 for their co-operativity with BCR-ABL revealed that AML1L419X and AML1H377X were able to co-operate with BCR-ABL to immortalize progenitor cells, while AML1I337X and AML1A297X did not. These findings suggest that transcriptional regulatory domains residing at the carboxyl-terminal of AML1/RUNX1 play important roles for wild-type AML1/RUNX1 to induce BC-like disease.Perhaps consistent with the increased expression of AML1/RUNX1 contributing to the occurrence of BC, shRNA-mediates silencing of AML1/RUNX1 inhibited growth of human CML cell lines (K562, MegA2, and MEG01).The overall findings of the present study suggest that (1) all mutants of AML1/RUNX1 do not contribute equally to the induction of BC-like disease, but there is a wide range of variance among mutants regarding their ability to induce BC in collaboration with BCR-ABL, and (2) increased expression of wild-type AML1/RUNX1 can induce BC-like disease in collaboration with BCR-ABL in mice. AML1/RUNX1 mutants tested here are known to dominant-negatively inhibit wild-type AML1/RUNX1 functions, but only two of 4 mutants exhibited BC-inducing activity. The over-expression of wild-type AML1/RUNX1 also exhibited such BC-inducing activity. We are currently conducting a detailed investigation of differences in cell lineage affected and growth properties caused by mutant and wild-type AML1/RUNX1s, which may reveal the dichotomic functions of AML1/RUNX1 in the development of BC. Disclosures:Naoe:Zenyaku-Kogyo: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Chugai Pharma.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Kyowa-Hakko Kirin.: Research Funding; Otsuka Pharma.: Research Funding.

A High Throughput Screening System for the Identification of Novel Genes in the Regulation of Mammalian Erythropoiesis

Abstract 2093Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursors. This induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. A recent genome wide transcriptional profile study demonstrated that over 600 genes are up-regulated during erythropoiesis. Surprisingly, the roles that most of the identified genes play in erythroid cells are still unknown. Understanding the functions of these unknown genes in the erythroid cells is necessary to elucidate the pathogenesis of red cell disorders such as congenital dyserythropoietic anemias, fanconi anemia, aplastic anemia, megaloblastic anemia, as well as leukemia and myelodysplastic syndromes with leukemic or dysplastic erythroid features. The goal of our study is to identify novel genes involved in different stages of erythropoiesis. To achieve this goal, we developed a high-throughput flow cytometry based assay that simultaneously detects erythroid cell differentiation and enucleation. In this assay, mouse fetal liver erythroblasts were purified and infected with lentiviruses containing a mammalian shRNA knockdown library obtained from the Broad Institute. The infected cells were cultured in a 96-well plate. Over the following two days the unaffected cells fully differentiate with approximately 60% of the cells enucleated. However, those cells in which shRNAs have knocked down genes critical for erythropoiesis are expected to show alterations in differentiation and/or enucleation. The system was validated using lentiviruses expressing shRNAs against Gata1 and mDia2, known proteins that are involved in the early and late stages of erythropoiesis, which showed inhibitions of differentiation and enucleation, respectively. We have pre-screened the most up-regulated 100 genes that play unknown functions in the erythroid cells. Future studies will be focused on the identified novel genes on their functions in erythroid lineage commitment, Epo mediated signaling pathways, hemoglobin enrichment, chromatin condensation, and enucleation. Disclosures:No relevant conflicts of interest to declare.

Oncogene Dosage Is a Major Determinant of Leukemogenic Transformation by AE9a

Abstract 1358Chromosomal translocation (8;21) results in the formation of the AML1-ETO (AE) leukemia-associated fusion protein. Roughly 70% of AE+ patient samples also harbor an alternatively spliced variant of AE, termed AE9a. AE9a promotes the leukemic transformation of mouse hematopoietic stem/progenitor cells without the need for secondary external cooperating oncogenes. By comparison, leukemia development in response to expression of full length AE requires one or more additional events. We extended these findings into a human cell system to determine whether AE9a is able to promote the transformation of human CD34+ cord blood (CB) cells to acute myeloid leukemia (AML). Transduction of CD34+ CB cells with AE9a promotes long-term culture potential, enhanced replating capacity, and expansion of CD34+ progenitor cells similar to full length AE. While we have not yet been able to generate AE9a leukemia in xenograft assays (despite several attempts), subcutaneous injection of AE9a cells promotes formation of a robust granulocytic sarcoma which is not seen using cells expressing full length AE. Several of our analyses have highlighted the importance of increased AE9a fusion protein expression in its enhanced function relative to full length AE, a concept that was also observed using the MLL-AF9 leukemia oncogene (Chen et. al, Cancer Cell, 2008). Indeed, flow cytometric sorting of freshly transduced AE or AE9a cultures based on high and low fusion protein expression revealed the importance of expression level for both AE and AE9a. High AE expression completely blocked colony formation and decreased cell expansion. These effects correlated with a dramatic upregulation of the cell cycle inhibitor p21. Conversely, high levels of AE9a did not upregulate p21 to the same degree, and colony formation and cell expansion were not as dramatically affected. In contrast, flow sort for cells expressing low levels of AE or AE9a had significantly less remarkable effects on cell function and p21 expression. Unexpectedly, AE9a-low cells eventually came to resemble the AE9a-high in terms of AE9a protein levels. These effects were not seen for full length AE sorted cultures. To determine whether similar effects were occurring in the murine AML model, we examined the effects of low and high expression levels of AE9a in mouse fetal liver cells. Corroborating our findings with the human cell model, AE9a-low samples promoted significantly less AML as compared to AE9a-high cells. This was not due to lack of engraftment of these cells or to loss of AE9a expression. Similar effects were found in vitro, where AE9a-low cells were unable to replate in methylcellulose assays while AE9a-high cells as well as AE-high and AE-low cells showed enhanced replating ability. Strikingly, the AML that did develop in mice transplanted with AE9a-low cells reverted to a very high level of AE9a protein expression, implying that increased expression of AE9a is essential for function. To better understand the mechanistic link between increased AE9a expression and AML development, we performed global gene expression analysis of murine progenitor cells expressing AE or AE9a (after three weeks of in vivo expansion). Analysis revealed a subset of genes that were highly repressed in AE9a expressing cells as compared to full length AE. Given that AE9a lacks the carboxy-terminal NHR3 and NHR4 domains of AE that interact with the NCoR and SMRT corepressors, the finding of significantly more repressed genes in AE9a cells is quite surprising. Network analysis has uncovered potential signaling pathways that may be involved in AE9a mediated transformation. Uncovering the mechanism of AE9a function could lead to a better understanding of t(8;21)-associated function in human AML. Disclosures:No relevant conflicts of interest to declare.

Ultrastructural study of transcription factories in mouse erythroblasts

RNA polymerase II (RNAPII) transcription has been proposed to occur at transcription factories; nuclear focal accumulations of the active, phosphorylated forms of RNAPII. The low ratio of transcription factories to active genes and transcription units suggests that genes must share factories. Our previous analyses using light microscopy have indicated that multiple genes could share the same factory. Furthermore, we found that a small number of specialized transcription factories containing high levels of the erythroid-specific transcription factor KLF1 preferentially transcribed a network of KLF1-regulated genes. Here we used correlative light microscopy in combination with energy filtering transmission electron microscopy (EFTEM) and electron microscopy in situ hybridization (EMISH) to analyse transcription factories, transcribing genes, and their nuclear environments at the ultrastructural level in ex vivo mouse foetal liver erythroblasts. We show that transcription factories in this tissue can be recognized as large nitrogen-rich structures with a mean diameter of 130 nm, which is considerably larger than that previously seen in transformed cultured cell lines. We show that KLF1-specialized factories are significantly larger, with the majority of measured factories occupying the upper 25th percentile of this distribution with an average diameter of 174 nm. In addition, we show that very highly transcribed genes associated with erythroid differentiation tend to occupy and share the largest factories with an average diameter of 198 nm. Our results suggest that individual factories are dynamically organized and able to respond to the increased transcriptional load imposed by multiple highly transcribed genes by significantly increasing in size.

Generation of Hematopoiesis-specific Cre Transgenic Mouse

EDAG is a specifically expressed gene of hematopoietic stem cell during embryo development. To obtain hematopoietic stem cell specific Cre recombinase transgenic mice, and construct conditional knocking-out target gene during early hematopoiesis, a targeting vector (EDAG-Cre) containing EDAG promoter and Cre recombinase gene was generated and linearized. By microinjecting 5.6 kb linearized EDAG-Cre into pronuclear fertilized oocyte, the pups were characterized by genomic PCR, and the conventional method was used for proliferation. The results show that six Cre positive transgenic mice were identified, and four of them were stably transferred. RT-PCR analysis shows that the Cre recombinase gene was expressed in bone marrow, spleen, thymus, peripheral blood, fetal liver and other tissues of the positive transgenic mice, and Cre recombinase activity in bone marrow and spleen were also confirmed. It demonstrates that EDAG-Cre transgenic mice were established and could be used for generating hematopoietic stem cell specific conditional knockout mice.

Transcription Factors KLF1 and KLF2 Positively Regulate Embryonic and Fetal β-Globin Genes through Direct Promoter Binding

Krüppel-like factors (KLFs) control cell differentiation and embryonic development. KLF1 (erythroid Krüppel-like factor) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β-globin genes. KLF1 and KLF2 have highly homologous zinc finger DNA-binding domains. They have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double KO mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, but double KO embryos are more anemic and die sooner than either single KO. In this work, a dual human β-globin locus transgenic and KLF knockout mouse model was used. The results demonstrate that the human ε- (embryonic) and γ-globin (fetal) genes are positively regulated by KLF1 and KLF2 in embryos. Conditional KO mouse experiments indicate that the effect of KLF2 on embryonic globin gene regulation is at least partly erythroid cell-autonomous. KLF1 and KLF2 bind directly to the promoters of the human ε- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region, as demonstrated by ChIP assays with mouse embryonic blood cells. H3K9Ac and H3K4me3 marks indicate open chromatin and active transcription, respectively. These marks are diminished at the Ey-, βh1-, ε- and γ-globin genes and locus control region in KLF1(-/-) embryos, correlating with reduced gene expression. Therefore, KLF1 and KLF2 positively regulate the embryonic and fetal β-globin genes through direct promoter binding. KLF1 is required for normal histone modifications in the β-globin locus in mouse embryos.

Embryonic Ductal Plate Cells Give Rise to Cholangiocytes, Periportal Hepatocytes, and Adult Liver Progenitor Cells

BACKGROUND& AIMS: Embryonic biliary precursor cells form a periportal sheet called the ductal plate, which is progressively remodeled to generate intrahepatic bile ducts. A limited number of ductal plate cells participate in duct formation; those not involved in duct development are believed to involute by apoptosis. Moreover, cells that express the SRY-related HMG box transcription factor 9 (SOX9), which include the embryonic ductal plate cells, were proposed to continuously supply the liver with hepatic cells. We investigated the role of the ductal plate in hepatic morphogenesis. Apoptosis and proliferation were investigated by immunostaining of mouse and human fetal liver tissue. The postnatal progeny of SOX9-expressing ductal plate cells was analyzed after genetic labeling, at the ductal plate stage, by Cre-mediated recombination of a ROSA26RYFP reporter allele. Inducible Cre expression was induced by SOX9 regulatory regions, inserted in a bacterial artificial chromosome. Livers were studied from mice under normal conditions and during diet-induced regeneration. Ductal plate cells did not undergo apoptosis and showed limited proliferation. They generated cholangiocytes lining interlobular bile ducts, bile ductules, and canals of Hering, as well as periportal hepatocytes. Oval cells that appeared during regeneration also derived from the ductal plate. We did not find that liver homeostasis required a continuous supply of cells from SOX9-expressing progenitors. The ductal plate gives rise to cholangiocytes lining the intrahepatic bile ducts, including its most proximal segments. It also generates periportal hepatocytes and adult hepatic progenitor cells.

The DNA binding factor Hmg20b is a repressor of erythroid differentiation

In erythroblasts, the CoREST repressor complex is recruited to target promoters by the transcription factor Gfi1b, leading to repression of genes mainly involved in erythroid differentiation. Hmg20b is a subunit of CoREST, but its role in erythropoiesis has not yet been established. To study the role of Hmg20b in erythropoiesis, we performed knockdown experiments in a differentiation-competent mouse fetal liver cell line, and in primary mouse fetal liver cells. The effects on globin gene expression were determined. We used microarrays to investigate global gene expression changes induced by Hmg20b knockdown. Functional analysis was carried out on Hrasls3, an Hmg20b target gene. We show that Hmg20b depletion induces spontaneous differentiation. To identify the target genes of Hmg20b, microarray analysis was performed on Hmg20b knockdown cells and controls. In line with its association to the CoREST complex, we found that 85% (527 out of 620) of the deregulated genes are up-regulated when Hmg20b levels are reduced. Among the few down-regulated genes was Gfi1b, a known repressor of erythroid differentiation. Among the consistently up-regulated targets were embryonic β-like globins and the phospholipase HRAS-like suppressor 3 (Hrasls3). We show that Hrasls3 expression is induced during erythroid differentiation and that knockdown of Hrasls3 inhibits terminal differentiation of proerythroblasts. We conclude that Hmg20b acts as an inhibitor of erythroid differentiation, through the down-regulation of genes involved in differentiation such as Hrasls3, and activation of repressors of differentiation such as Gfi1b. In addition, Hmg20b suppresses embryonic β-like globins.

Long Range Interactions Regulate Igf2 Gene Transcription during Skeletal Muscle Differentiation

The differentiation, maintenance, and repair of skeletal muscle is controlled by interactions between genetically determined transcriptional programs regulated by myogenic transcription factors and environmental cues activated by growth factors and hormones. Signaling through the insulin-like growth factor 1 (IGF1) receptor by locally produced IGF2 defines one such pathway that is critical for normal muscle growth and for regeneration after injury. IGF2 gene and protein expression are induced as early events in muscle differentiation, but the responsible molecular mechanisms are unknown. Here we characterize a distal DNA element within the imprinted mouse Igf2-H19 locus with properties of a muscle transcriptional enhancer. We find that this region undergoes a transition to open chromatin during differentiation, whereas adjacent chromatin remains closed, and that it interacts in differentiating muscle nuclei but not in mesenchymal precursor cells with the Igf2 gene found more than 100 kb away, suggesting that chromatin looping or sliding to bring the enhancer in proximity to Igf2 promoters is also an early event in muscle differentiation. Because this element directly stimulates the transcriptional activity of an Igf2 promoter-reporter gene in differentiating myoblasts, our results indicate that we have identified a bona fide distal transcriptional enhancer that supports Igf2 gene activation in skeletal muscle cells. Because this DNA element is conserved in the human IGF2-H19 locus, our results further suggest that its muscle enhancer function also is conserved among different mammalian species.

HEBAlt enhances the T-cell potential of fetal myeloid-biased precursors

Hematopoiesis is controlled by the interplay between transcription factors and environmental signals. One of the primary determinants of the T-lineage choice is Delta-like (DL)-Notch signaling, which promotes T-cell development and inhibits B-cell development. We have found that the transcription factor HEBAlt is up-regulated in early hematopoietic precursors in response to DL-Notch signaling and that it can promote early T-cell development. Here, we identified a population of lineage-negative Sca-1⁻c-kit(+) (LK) cells in the mouse fetal liver that rapidly gave rise to myeloid cells and B cells but exhibited very little T-cell potential. However, forced expression of HEBAlt in these precursors restored their ability to develop into T cells. We also showed that Ikaros and Notch1 are up-regulated in response to HEBAlt over-expression and that activated Notch1 enhances the ability of LK cells to enter the T-cell lineage. Furthermore, the myeloid transcription factor C/EBPα is down-regulated in response to HEBAlt. We therefore propose that HEBAlt plays a role in the network that enforces the T-lineage fate and limits myeloid fate during hematopoiesis.

A transmembrane glycoprotein, gp38, is a novel marker for immature hepatic progenitor cells in fetal mouse livers

Previously, we clarified the surface antigen profiles of hepatic progenitor cells (HPCs) in fetal liver tissue as the CD49f+CD45−Thy1− cell fraction. However, these cells were a heterogeneous cell population containing various stages of differentiation. This study aimed to detect more immature HPCs, using a novel surface antigen, gp38. After the collagenase digestion of fetal livers harvested from E13.5 to E18.5 fetal mice, HPCs were obtained and divided into two subpopulations using flow cytometry: gp38-positive HPCs, and gp38-negative HPCs. Both types of HPCs were characterized by immunocytochemistry and RT-PCR. The proliferative activity was compared by BrdU incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay. Furthermore, the comprehensive gene expression was investigated by DNA microarray. Both types of HPCs expressed alpha-fetoprotein. However, the gp38-positive HPCs derived from E13.5 fetal livers did not express albumin or cytokeratin 19, while the gp38-negative HPCs did. DNA microarray revealed that some genes related to the Wnt signal pathway were up-regulated in the gp38-positive HPCs. Furthermore, Wnt3a had a proliferative effect on the gp38-positive HPCs. In conclusion, the gp38-positive HPCs derived from fetal liver tissue until E13.5 could therefore be candidates for hepatic stem cells in the fetal liver.

Studies on Heme Oxygenase 1 During Erythroid Differentiation

AbstractAbstract 4254Heme is indispensable for the function of all aerobic cells as a prosthetic group of innumerable proteins. However, “free heme” (uncommitted) can initiate the formation of free radicals and cause lipid peroxidation, which can lead to cellular damage and tissue injury. Therefore, the rate of heme biosynthesis and catabolism must be well balanced by tight control mechanisms. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC), whose precursors synthesize heme with rates that are at least one order of magnitude higher (on the per cell basis) than those in the liver – the second most active heme producer in the body. The degradation of heme is exclusively carried out by heme oxygenases 1 and 2 (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using both a murine erythroleukemia cell line (MEL) and primary erythroid cells isolated from mouse fetal livers, we have demonstrated that during erythroid differentiation HO1 is up-regulated at both mRNA and protein levels. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase. These data suggest that in developing RBC, in addition to the continuous assembly of heme with globin chains, there is an increase in levels of uncommitted heme, which upregulates HO1 expression. Additionally, we have shown that down-regulation of HO1 via siRNA increased hemoglobinization in differentiating MEL cells. In contrast, induction of HO1 expression by NaAsO2 reduced the hemoglobinization of MEL cells. This effect could be reversed to control levels by the addition of HO1 inhibitor tin-protophorphyrin (SnPP). These results show that in differentiating erythroid cells the balance between levels of heme and HO1 have to be tightly regulated to maintain hemoglobinization at appropriate levels. Our results lead us to propose that disturbances in HO1 expression could play a role in some pathophysiological conditions such as thalassemias. Disclosures:No relevant conflicts of interest to declare.