Mammalian Hematopoiesis Research Articles

Evidence for the conservation of miR-223 in zebrafish (Danio rerio): Implications for function

MicroRNAs (miRNAs) are an abundant and conserved class of small RNAs, which play important regulatory functions by interacting with the 3′ untranslated region (UTR) of target mRNAs. Through this mechanism, miR-223 was shown to regulate genes involved in mammalian haematopoiesis, both in physiological and pathological contexts. MiR-223 is essential for normal myelopoiesis in mammals, promoting granulocyte, osteoclast and megakaryocyte differentiation and suppressing erythropoiesis. However, there is a general lack of knowledge regarding miR-223 function in other vertebrates, which could help to clarify its role in other processes, such as development. In this work, we explored the functional conservation of miR-223 using zebrafish as a model. We show that miR-223 gene structure and genomic context have been maintained between human and zebrafish. In addition, we identified 22 novel sequences of miR-223 precursor and demonstrate that it contains domains highly conserved among vertebrates, suggesting function preservation throughout evolution. Furthermore, collected evidences show that miR-223 expression is highly correlated with haematopoietic events and osteoclastogenesis throughout zebrafish development. In adults, expression of miR-223 in zebrafish tissues mimics the distribution in mice, with high levels found in the major fish haematopoietic organ, the head kidney. These results suggest a conservation of miR-223 role in haematopoiesis, and osteoclastogenesis between zebrafish and human. Accordingly, validated targets of miR-223 in mammalian models were investigated and defined as putative targets in zebrafish, by in silico and gene expression analysis. Our data compiles critical evidence showing that miR-223, a highly conserved miRNA, appears to have kept similar regulatory functions throughout evolution.

Detection of Rearrangements of Mixed Lineage Leukemia Gene and Its clinical relevance

Background: The mixed lineage leukemia (MLL) gene located on chromosome 11 band q23 normally functions as a transcription regulator of the HOX genes and is essential for normal mammalian development and hematopoiesis. Chromosomal translocations involving MLL gene represent frequent cytogenetic abnormalities found in aggressive acute leukemia, both lymphoblastic and myeloid. AL MLL rearrangements have the unique clinical, hematological and prognostic features. We aimed to study the incidence and the types of fusion genes and the clinical relevance.Results: Study samples were from 60 acute leukemia (AL) patients from Sep. 2003 to Dec. 2005. There were 28 males and 32 females. The ages were from 3 months to 54 years old. 17 patients were less than 15 years old and 43 patients were more than 15 years old. All patients were diagnosed based on FAB diagnostic criteria. The patients included 30 with ALL (10 of ALL-L1 patients, 15 of ALL-L2, 5 of ALL-L3), 28 with AML (2 of AML-M1 patients, 5 of AML-M2, 5 of AML-M4, 13 of AML-M5, 1 of AML-M6, 2 of AML-M7), 1 with mixed cell leukemia (AMLL), and 1 with NK cell leukemia. 59 patients were newly diagnosed and 1 patient was refractory. The rearrangements of MLL gene were detected by fluorescence in situ hybridization (FISH) and 6 types of common fusion genes (MLL / AF4, MLL / ENL, MLL / AF9, MLL / ELL MLL / AF6, MLL / AF10) resulting from the rearrangements of MLL gene were detected by nested RT-PCR. There arrangements of MLL gene was found in 7 out of 60 AL patients, (11.67%). Among these 7 patients, 2 were diagnosed with AML- M5 and 5 patients were diagnosed with B-ALL. The fusion genes of the 2 AML-M5 patients who had the rearrangements of MLL gene were MLL/AF9. Among 5 B-ALL patients, 2 patients were confirmed to express MLL/ENL, 1 patient was confirmed to express MLL/AF4, and the other 2 patients did not express the fusion genes. 1 of 2 AML-M5 MLL fusion gene positive patients had invasion of leukemia cell in the left leg and CR was achieved after the first course of chemotherapy. The central nervous system leukemia was got after 1 year and died. The other achieved CR with the third of chemotherapy. 1 of 5 patients died from DIC and the cerebral hemorrhage on the second day after diagnosis B-ALL of MLL fusion gene positive. 2 of 5 patients were not in remission and died from multi-organ failures and infection 3 weeks after diagnosis with positive MLL fusion gene. 1 of 5 patients B-ALL of MLL fusion gene positive got invasion of leukemia cell in thoracic and achieved CR after the second course of chemotherapy. 1 of 5 patients of B-ALL of MLL fusion gene positive achieved CR after the first course of chemotherapy and was relapse 1 year after the bone marrow transplant and then died.Conclusions: We conclude that nested RT-PCR is convenient and feasible method to detect the types of fusion genes resulting from the rearrangements of MLL gene. The clinical features of AL with MLL fusion gene include high white blood cell, invasion of multiple organs, resistant to conventional chemotherapy, easy to relapse after remission, and poor prognosis. The detection of MLL gene rearrangement is of great importance in predicting prognosis and guiding therapy in AL. DisclosuresNo relevant conflicts of interest to declare.

Stem cell health and tissue regeneration in microgravity.

Exposure to microgravity causes significant mechanical unloading of mammalian tissues, resulting in rapid alterations of their physiology, which poses a significant risk for long-duration manned spaceflight. The immediate degenerative effects of spaceflight we understand best are those studied during short-term low-Earth-orbit experiments, and include rapid microgravity-adaptive bone and muscle loss, loss of cardiovascular capacity, defects in wound and bone fracture healing, and impaired immune function. Over the long-term, exposure to microgravity may cause severe deficits in mammalian stem cell-based tissue regenerative health, including, osteogenesis, hematopoiesis, and lymphopoeisis, as well as cause significant stem cell-based tissue degeneration in amphibian tail and lens regeneration. To address the needs for stem cell and other cell science research on the International Space Station (ISS), NASA has developed the new Bioculture System that will allow investigators to initiate and conduct on-orbit experiments that astronauts will be able to monitor and interact with during the course of cell cultures. This cell culture capability combined with advanced technologies for molecular biology and on-orbit measurement of gene expression (WetLab2) and other tools that are now coming online bring the ISS National Laboratory a step closer to becoming a fully functional space laboratory for advancing space biological sciences.

Modeling murine yolk sac hematopoiesis with embryonic stem cell culture systems

The onset of hematopoiesis in mammals is defined by generation of primitive erythrocytes and macrophage progenitors in embryonic yolk sac. Laboratories have met the challenge of transient and swiftly changing specification events from ventral mesoderm through multipotent progenitors and maturing lineage-restricted hematopoietic subtypes, by developing powerful in vitro experimental models to interrogate hematopoietic ontogeny. Most importantly, studies of differentiating embryonic stem cell derivatives in embryoid body and stromal coculture systems have identified crucial roles for transcription factor networks (e.g. Gata1, Runx1, Scl) and signaling pathways (e.g. BMP, VEGF, WNT) in controlling stem and progenitor cell output. These and other relevant pathways have pleiotropic biological effects, and are often associated with early embryonic lethality in knockout mice. Further refinement in subsequent studies has allowed conditional expression of key regulatory genes, and isolation of progenitors via cell surface markers (e.g. FLK1) and reporter-tagged constructs, with the purpose of measuring their primitive and definitive hematopoietic potential. These observations continue to inform attempts to direct the differentiation, and augment the expansion, of progenitors in human cell culture systems that may prove useful in cell replacement therapies for hematopoietic deficiencies. The purpose of this review is to survey the extant literature on the use of differentiating murine embryonic stem cells in culture to model the developmental process of yolk sac hematopoiesis.

Intrinsic and extrinsic regulation of mammalian hematopoiesis in the fetal liver.

The fetal liver (FL) is an important structure in expansion and differentiation of hematopoietic stem cells (HSC), but despite this little is known about the exact mechanisms in which FL hematopoiesis takes place. Primitive hematopoiesis gives way to definitive hematopoiesis at 12.5 dpc in mice and the process is regulated by a number of intrinsic and extrinsic factors. Intrinsic regulations are intracellular processes that have been reported to be important in the initiation of definitive hematopoiesis. Several structures are involved with extrinsic regulations of hematopoiesis within the FL, including hepatoblasts and liver sinusoidal endothelial cells (LSEC). Hepatoblasts and endothelial cells comprise separate niches involved in the extrinsic regulation of hematopoiesis. Studies have shown that co-cultures with fetal liver stromal cells can promote the expansion of erythroid cells, although the way in which stromal cells do this is still unknown. Understanding the mechanisms in which hematopoiesis is regulated in the FL could lead to the production of novel therapies involving the safe and reliable transplantation of HSCs to patients with blood and bone marrow complications. This review aims to summarize the current state of knowledge about the regulation of hematopoiesis specifically within the FL.

Lymphomyeloid Contribution of an Immune-Restricted Progenitor Emerging Prior to Definitive Hematopoietic Stem Cells

In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis.

Regulation of MEIS1 by distal enhancer elements in acute leukemia

Aberrant activation of the three-amino-acid-loop extension (TALE) homeobox gene MEIS1 shortens the latency and accelerates the onset and progression of acute leukemia, yet the molecular mechanism underlying persistent activation of the MEIS1 gene in leukemia remains poorly understood. Here we used a combined comparative genomics analysis and an in vivo transgenic zebrafish assay to identify 6 regulatory DNA elements that are able to direct GFP expression in a spatiotemporal manner during zebrafish embryonic hematopoiesis. Analysis of chromatin characteristics and regulatory signatures suggest that many of these predicted elements are potential enhancers in mammalian hematopoiesis. Strikingly, one of the enhancer elements (E9) is a frequent integration site in retroviral induced mouse acute leukemia. The genomic region corresponding to enhancer E9 is differentially marked by H3K4 mono-methylation and H3K27 acetylation, hallmarks of active enhancers, in multiple leukemia cell lines. Decreased enrichment of these histone marks is associated with downregulation of MEIS1 expression during hematopoietic differentiation. Furthermore, MEIS1/HOXA9 transactivate this enhancer via a conserved binding motif in vitro, and participate in an autoregulatory loop that modulates MEIS1 expression in vivo. Our results suggest that an intronic enhancer regulates the expression of MEIS1 in hematopoiesis and contributes to its aberrant expression in acute leukemia.

A role for RUNX1 in hematopoiesis and myeloid leukemia

Since its discovery from a translocation in leukemias, the runt-related transcription factor 1/acute myelogenous leukemia-1 (RUNX1/AML1), which is widely expressed in hematopoietic cells, has been extensively studied. Many lines of evidence have shown that RUNX1 plays a critical role in regulating the development and precise maintenance of mammalian hematopoiesis. Studies using knockout mice have shown the importance of RUNX1 in a wide variety of hematopoietic cells, including hematopoietic stem cells and megakaryocytes. Recently, target molecular processes of RUNX1 in normal and malignant hematopoiesis have been revealed. Although RUNX1 is not required for the maintenance of hematopoietic stem cells, it is required for the homeostasis of hematopoietic stem and progenitor cells, and expansion of hematopoietic stem and progenitor cells due to RUNX1 deletion may be an important cause of human leukemias. Molecular abnormalities cooperating with loss of RUNX1 have also been identified. These findings may lead to a further understanding of human leukemias, and suggest novel molecular targeted therapies in the near future.

Ginger stimulates hematopoiesis via Bmp pathway in zebrafish.

BackgroundAnemia is a hematologic disorder with decreased number of erythrocytes. Erythropoiesis, the process by which red blood cells differentiate, are conserved in humans, mice and zebrafish. The only known agents available to treat pathological anemia are erythropoietin and its biologic derivatives. However, erythropoietin therapy elicits unwanted side-effects, high cost and intravenous or subcutaneous injection, warranting the development of a more cost effective and non-peptide alternative. Ginger (Zingiber officinale) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis.Methodology/Principal FindingsHere, we utilized gata1:dsRed transgenic zebrafish embryos to investigate the effect of ginger extract on hematopoiesis in vivo and we identified its bioactive component, 10-gingerol. We confirmed that ginger and 10-gingerol promote the expression of gata1 in erythroid cells and increase the expression of hematopoietic progenitor markers cmyb and scl. We also demonstrated that ginger and 10-gingerol can promote the hematopoietic recovery from acute hemolytic anemia in zebrafish, by quantifying the number of circulating erythroid cells in the dorsal aorta using video microscopy. We found that ginger and 10-gingerol treatment during gastrulation results in an increase of bmp2b and bmp7a expression, and their downstream effectors, gata2 and eve1. At later stages ginger and 10-gingerol can induce bmp2b/7a, cmyb, scl and lmo2 expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1.Conclusions/SignificanceOur study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents.

Modulation of Wnt5a Expression by Periodontopathic Bacteria

Wingless proteins, termed Wnt, are involved in embryonic development, blood cell differentiation, and tumorigenesis. In mammalian hematopoiesis, Wnt signaling is essential for stem-cell homeostasis and lymphocyte differentiation. Recent studies have suggested that these molecules are associated with cardiovascular diseases, rheumatoid arthritis, and osteoarthritis. Furthermore, Wnt5a signaling is essential for the general inflammatory response of human macrophages. Periodontitis is a chronic inflammatory disease caused by gram-negative periodontopathic bacteria and the resultant host immune response. Periodontitis is characterized by loss of tooth-supporting structures and alveolar bone resorption. There have been no previous reports on Wnt5a expression in periodontitis tissue, and only few study reported the molecular mechanisms of Wnt5a expression in LPS-stimulated monocytic cells. Using RT-PCR, we demonstrated that Wnt5a mRNA expression was up-regulated in chronic periodontitis tissue as compared to healthy control tissue. P. gingivalis LPS induced Wnt5a mRNA in the human monocytic cell line THP-1 with a peak at 4 hrs after stimulation. P. gingivalis LPS induced higher up-regulation of Wnt5a mRNA than E. coli LPS. The LPS receptors TLR2 and TLR4 were equally expressed on the surface of THP-1 cells. P. gingivalis LPS induced IκBα degradation and was able to increase the NF-κB binding activity to DNA. P. gingivalis LPS-induced Wnt5a expression was inhibited by NF-κB inhibitors, suggesting NF-κB involvement. Furthermore, IFN-γ synergistically enhanced the P. gingivalis LPS-induced production of Wnt5a. Pharmacological investigation and siRNA experiments showed that STAT1 was important for P. gingivalis LPS-induced Wnt5a expression. These results suggest that the modulation of Wnt5a expression by P. gingivalis may play an important role in the periodontal inflammatory process and serve a target for the development of new therapies.

Size control of the Drosophila hematopoietic niche by bone morphogenetic protein signaling reveals parallels with mammals

The Drosophila melanogaster larval hematopoietic organ, the lymph gland, is a model to study in vivo the function of the hematopoietic niche. A small cluster of cells in the lymph gland, the posterior signaling center (PSC), maintains the balance between hematopoietic progenitors (prohemocytes) and their differentiation into specialized blood cells (hemocytes). Here, we show that Decapentaplegic/bone morphogenetic protein (Dpp/BMP) signaling activity in PSC cells controls niche size. In the absence of BMP signaling, the number of PSC cells increases. Correlatively, no hemocytes differentiate. Controlling PSC size is, thus, essential for normal blood cell homeostasis. Activation of BMP signaling in the PSC requires expression of the Dally-like heparan-sulfate proteoglycan, under the control of the Collier/early B-cell factor (EBF) transcription factor. A Dpp > dpp autoregulatory loop maintains BMP signaling, which limits PSC cell proliferation by repressing the protooncogene dmyc. Dpp antagonizes activity of wingless (Wg)/Wnt signaling, which positively regulates the number of PSC cells via the control of Dmyc expression. Together, our data show that Collier controls hemocyte homeostasis via coordinate regulation of PSC cell number and PSC signaling to prohemocytes. In mouse, EBF2, BMP, and Wnt signaling in osteoblasts is required for the proper number of niche and hematopoietic stem cells. Our findings bring insights to niche size control and draw parallels between Drosophila and mammalian hematopoiesis.

The critical role of histone H2A-deubiquitinase Mysm1 in hematopoiesis and lymphocyte differentiation

AbstractStem cell differentiation and lineage specification depend on coordinated programs of gene expression, but our knowledge of the chromatin-modifying factors regulating these events remains incomplete. Ubiquitination of histone H2A (H2A-K119u) is a common chromatin modification associated with gene silencing, and controlled by the ubiquitin-ligase polycomb repressor complex 1 (PRC1) and H2A-deubiquitinating enzymes (H2A-DUBs). The roles of H2A-DUBs in mammalian development, stem cells, and hematopoiesis have not been addressed. Here we characterized an H2A-DUB targeted mouse line Mysm1tm1a/tm1a and demonstrated defects in BM hematopoiesis, resulting in lymphopenia, anemia, and thrombocytosis. Development of lymphocytes was impaired from the earliest stages of their differentiation, and there was also a depletion of erythroid cells and a defect in erythroid progenitor function. These phenotypes resulted from a cell-intrinsic requirement for Mysm1 in the BM. Importantly, Mysm1tm1a/tm1a HSCs were functionally impaired, and this was associated with elevated levels of reactive oxygen species, γH2AX DNA damage marker, and p53 protein in the hematopoietic progenitors. Overall, these data establish a role for Mysm1 in the maintenance of BM stem cell function, in the control of oxidative stress and genetic stability in hematopoietic progenitors, and in the development of lymphoid and erythroid lineages.

DNA Methylation Profile of Runx1 Regulatory Regions Is Correlated with Transition From Primitive to Definitive Hematopoietic Potential In Vitro and In Vivo

Abstract 389 Introduction:The Runt-related transcription factor Runx1 (AML1) is a central regulator of mammalian hematopoiesis and is required for the generation of hematopoietic stem cells (HSC) from hemogenic endothelium in the embryo. It has been shown that Runx1 is alternatively expressed from two promoters in a temporal fashion, and that their differential activities are influenced by a conserved intronic enhancer (+23) element. Intriguingly, promoter usage follows a pattern whereby the proximal (P2) initiates early in primitive hematopoiesis, while the distal (P1) becomes active later at the time of HSC emergence and is the predominant isoform expressed in fetal liver and adult HSC. While some transcription factor binding sites and cis-regulatory elements have been identified, an explanation for the alternative promoter usage remains elusive. We hypothesized that this regulation may be at the level of chromatin accessibility, and therefore investigated the DNA methylation status of Runx1 cis-elements. Methods/Results:We analyzed bisulfite-treated genomic DNA from E14.5 fibroblast (MEF), E8.5 yolk sac CD41+ (YS), E14.5 fetal liver Lin-Sca-1+CD48-CD150+ (FL), and adult marrow Lin-cKit+Sca-1+ (KLS); representing non-hematopoietic, primitive hematopoietic, and two stages of definitive HSC respectively. In addition, we also examined methylation in hematopoietic populations derived in vitro from murine embryonic stem cells (mESC). Initial exploratory analysis focused on classically defined CpG islands upstream of each promoter, however no significant differential methylation was observed within these regions. Subsequent analysis focused on CpGs near the transcription start site (TSS) and within the +23 enhancer. The P2 promoter was uninformative as it was unmethylated in all populations analyzed, whereas methylation within the +23 enhancer differentiated between hematopoietic and non-hematopoietic cell populations. At the P1 promoter, methylation status was remarkably correlated with primitive vs. definitive status. P1 was highly methylated in MEFs (77%), mESC embryoid body (EB) derived cKit+CD41+ (66%), and E8.5 YS CD41+ (58%); but significantly less methylated in vivo in FL HSC (8.1%) and adult KLS cells (18%). We are currently using this correlation of demethylation and definitive HSC potential to identify conditions that may drive definitive HSC generation from mESC-derived blood progenitors. Since overexpression of HoxB4 coupled with OP9 co-culture is the only confirmed method capable of producing definitive HSC from mESC, and HoxB4 has been shown to bind within the P1 promoter region of Runx1, we cultured HoxB4 or control EB-derived hematopoietic progenitors on OP9 stroma. We observed progressive demethylation in the HoxB4 arm: after 6 days of co-culture 47% vs. 71% in controls, and after 11 days 27% in the HoxB4 arm while the control population failed to proliferate past day 6. Isoform specific RT-PCR confirmed that HoxB4 overexpression resulted in Runx1 expression from the P1 promoter whereas the control vector did not. Within P1, we identify a single CpG that is most highly correlated with definitive HSC potential in vivo, and most significantly demethylated upon HoxB4 overexpression in vitro. Conclusions:These data indicate that differential methylation occurs at Runx1 regulatory regions during hematopoietic development in vitro and in vivo. The +23 enhancer is demethylated in cells with hematopoietic potential, whereas demethylation of the Runx1 P1 promoter is highly correlated with definitive HSPC populations and is promoted in vitro by HoxB4. These data are the first to identify a role for DNA methylation in the regulation of alternative promoter usage at the Runx1 locus, and may serve as a novel biomarker of HSC potential during embryonic development. Disclosures:No relevant conflicts of interest to declare.

Zebrafish mll Gene Is Essential for Hematopoiesis

Studies implicate an important role for the mixed lineage leukemia (Mll) gene in hematopoiesis, mainly through maintaining Hox gene expression. However, the mechanisms underlying Mll-mediated hematopoiesis during embryogenesis remain largely unclear. Here, we investigate the role of mll during zebrafish embryogenesis, particularly hematopoiesis. Mll depletion caused severe defects in hematopoiesis as indicated by a lack of blood flow and mature blood cells as well as a significant reduction in expression of hematopoietic progenitor and mature blood cell markers. Furthermore, mll depletion prevented the differentiation of hematopoietic progenitors. In addition, we identified the N-terminal mini-peptide of Mll that acted as a dominant negative form to disrupt normal function of mll during embryogenesis. As expected, mll knockdown altered the expression of a subset of Hox genes. However, overexpression of these down-regulated Hox genes only partially rescued the blood deficiency, suggesting that mll may target additional genes to regulate hematopoiesis. In the mll morphants, microarray analysis revealed a dramatic up-regulation of gadd45αa. Multiple assays indicate that mll inhibited gadd45αa expression and that overexpression of gadd45αa mRNA led to a phenotype similar to the one seen in the mll morphants. Taken together, these findings demonstrate that zebrafish mll plays essential roles in hematopoiesis and that gadd45αa may serve as a potential downstream target for mediating the function of mll in hematopoiesis.

Self-renewal in late-stage erythropoiesis

Extensive in vitro self-renewal of proerythroblasts from the earliest period of definitive erythropoiesis, as reported by England and co-investigators in this issue of Blood, expands the potential role of self-renewal in mammalian hematopoiesis and suggests a possible source of blood cells for clinical treatments.

Activation of a Novel Palmitoyltransferase through a t(6;14) Translocation In T/Myeloid Biphenotypic Leukemia and Subsets of Acute Myeloid Leukemia.

AbstractAbstract 1663The current World Health Organization (WHO) classification of hematologic malignancies includes a number of entities defined by specific genetic abnormalities. Most known abnormalities result from chromosomal translocations and mutations involving genes encoding transcription factors or tyrosine kinases. We identified the activation of a novel palmitoyltransferase, ZDHHC14, through a chromosomal translocation, t(6;14)(q25;q32). This translocation was initially documented by three independent groups in two children and two adult patients with T/myeloid biphenotypic leukemia, a poorly differentiated form of acute leukemia with a single population of leukemic blasts co-expressing antigens of both T-lymphoid and myeloid lineages. Recently, we detected the same translocation in two additional patients with T/myeloid biphenotypic leukemia. Molecular characterization of the translocation breakpoints revealed the juxtaposition of a regulatory region downstream of BCL11B (at chromosome region 14q32) upstream of an uncharacterized gene, ZDHHC14 (at chromosome region 6q25), leading to transcriptional activation of the ZDHHC14. Notably, this regulatory region from 14q32 has been found to be responsible for transcriptional activation of a homeobox gene, TLX3, as a result of a cryptic translocation t(5;14)(q35;q32) in T-cell acute lymphoblastic leukemia. Our subsequent survey of ZDHHC14 protein levels in blasts from patients with various subtypes of acute myeloid leukemia (AML) showed that the protein was not detectable in AML patients with t(8;21), t(15;17), or inv(16), but was detected in AML patients with FLT3 mutations or with complex karyotypes, indicating a possible association of increased expression of this protein with unfavorable prognostic features of the disease. We also demonstrated that overexpression of ZDHHC14 could inhibit TPA-induced cellular differentiation of K562 cells. Interestingly, the Drosophila homolog of ZDHHC14 has been found to be a palmitoyltransferase regulating Fat signaling. Fat is a Drosophila tumor suppressor gene, encoding a large cadherin that regulates growth and a form of tissue organization. While the function of ZDHHC14 and the role of protein palmitoylation in mammalian hematopoiesis remain unknown, recent studies in neuronal development have demonstrated that palmitoylation regulates diverse aspects of protein trafficking. Our findings suggest that palmitoyltransferase plays a previously unappreciated role in hematopoiesis and that increased expression of ZDHHC14 may be associated with a primitive status of leukocytes. Further characterization of ZDHHC14 may broaden our knowledge of leukemogenesis, contribute to risk stratification of acute leukemia, and provide a novel therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Temporal specification of blood progenitors from mouse embryonic stem cells and induced pluripotent stem cells

The efficient and reproducible generation of differentiated progenitors from pluripotent stem cells requires the recapitulation of appropriate developmental stages and pathways. Here, we have used the combination of activin A, BMP4 and VEGF under serum-free conditions to induce hematopoietic differentiation from both embryonic and induced pluripotent stem cells, with the aim of modeling the primary sites of embryonic hematopoiesis. We identified two distinct Flk1-positive hematopoietic populations that can be isolated based on temporal patterns of emergence. The earliest arising population displays characteristics of yolk sac hematopoiesis, whereas a late developing Flk1-positive population appears to reflect the para-aortic splanchnopleura hematopoietic program, as it has reduced primitive erythroid capacity and substantially enhanced myeloid and lymphoid potential compared with the earlier wave. These differences between the two populations are accompanied by differences in the expression of Sox17 and Hoxb4, as well as in the cell surface markers AA4.1 and CD41. Together, these findings support the interpretation that the two populations are representative of the early sites of mammalian hematopoiesis.

Cdx4 is dispensable for murine adult hematopoietic stem cells but promotes MLL-AF9-mediated leukemogenesis

Cdx4 is a homeobox gene essential for normal blood formation during embryonic development in the zebrafish, through activation of posterior Hox genes. However, its role in adult mammalian hematopoiesis has not been extensively studied and its requirement in leukemia associated with Hox gene expression alteration is unclear. We inactivated Cdx4 in mice through either a germline or conditional knockout approach and analyzed requirement for Cdx4 in both normal adult hematopoiesis and leukemogenesis initiated by the MLL-AF9 fusion oncogene. Here, we report that loss of Cdx4 had a minimal effect on adult hematopoiesis. Indeed, although an increase in white blood cell counts was observed, no significant differences in the distribution of mature blood cells, progenitors or stem cells were observed in Cdx4-deficient animals. In addition, long-term repopulating activity in competitive transplantation assays was not significantly altered. In vitro, B-cell progenitor clonogenic potential was reduced in Cdx4-deficient animals but no significant alteration of mature B cells was detected in vivo. Finally, induction of acute myeloid leukemia in mice by MLL-AF9 was significantly delayed in the absence of Cdx4 in a retroviral transduction/bone marrow transplant model. These observations indicate that Cdx4 is dispensable for the establishment and maintenance of normal hematopoiesis in adult mammals. These results, therefore, outline substantial differences in the Cdx-Hox axis between mammals and zebrafish and support the hypothesis that Cdx factors are functionally redundant during mammalian hematopoietic development under homeostatic conditions. In addition, our results suggest that Cdx4 participates in MLL-AF9-mediated leukemogenesis supporting a role for Cdx factors in the pathogenesis of myeloid leukemia.

P89c‐Myb is not required for fetal or adult hematopoiesis

The c-myb gene encodes two proteins, termed p75 and p89. Of these, the larger isoform is transcribed from an alternatively spliced message that contains an additional exon, exon 9A. Disruption of the c-myb locus in mice results in embryonic lethality due to defective hematopoiesis and in the adult, tissue-specific inactivation of c-myb in hematopoietic tissues blocks differentiation along several lineages. The c-myb knock-out mouse models described thus far result in the disruption of both the p75 and p89 isoforms, making it impossible to assign a definitive role to p89(c-Myb) in development and hematopoiesis. We have therefore generated a null-mutant mouse where exon 9A has been systemically deleted that results in the absence of only the p89-myb transcript and protein. Unlike disruption of both forms of the c-myb gene, loss of only the p89-encoding isoform does not have any deleterious effects on mammalian hematopoiesis and development.

A Genetic Strategy for Single and Combinatorial Analysis of miRNA Function in Mammalian Hematopoietic Stem Cells

The regulatory role of micro-RNAs (miRNAs) in hematopoietic development is increasingly appreciated. Reverse genetics strategies based on the targeted disruption of miRNAs offer a powerful tool to study miRNA functions in mammalian hematopoiesis. The miR-144/451 cluster comprises two miRNAs coexpressed from a common precursor transcript in an erythroid-specific manner. To decipher the contribution of each miRNA of the cluster in mammalian erythropoiesis, we developed a strategy for stable in vivo individual and combinatorial miRNA inhibition. We developed decoy target sequences for each miRNA expressed by lentiviral vectors marked with distinct fluorescent proteins and used them to probe the functions of miR-144 and miR-451 in the murine hematopoietic system in a competitive repopulation setting. Murine hematopoietic chimeras expressing lentiviral-encoded inhibitory sequences specific for miR-144 or miR-451 exhibited markedly reduced Ter119(+) erythroblast counts, with the combined knockdown showing additive effect. These chimeras showed abnormal patterns of erythroid differentiation primarily affecting the proerythroblast to basophilic erythroblast transition, coinciding with the stage where expression of the miRNA cluster is dramatically induced and posttranscriptional gene regulation becomes prominent. These results reveal a role for the miR-144/451 locus in mammalian erythropoiesis and provide the first evidence of functional cooperativity between clustered miRNAs in the hematopoietic system. The strategy described herein will prove useful in functional miRNA studies in mammalian hematopoietic stem cells.