Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

LMO2 expression correlated with increased response to olaparib and olaparib with R-CHOP.

Transcription of the platelet-derived growth factor receptor α (PDGFRA/Pdgfra) gene is considered to be precisely regulated. We have previously reported that the PDGFRA/Pdgfra gene is regulated by a dual promoter system in human and mouse, in which a novel PDGFRA/Pdgfra transcript has a first exon (exon 1β) different from that of the canonical PDGFRA/Pdgfra transcript (exon 1α). To elucidate the function of each transcript, we first investigated the contribution of different PDGFRA transcripts to final protein levels. Notably, knockdown experiments suggested the existence of other PDGFRA transcripts, and we identified five additional first exons (exons 1γ, 1δ, 1ε, 1ζ, and 1η) in intron 1 in both the human and mouse genes. The first exons of the mouse Pdgfra gene showed unique expression patterns: exon 1α was broadly expressed; exon 1β was highly expressed in embryos; exon 1γ was observed at relatively high levels in the adult central nervous system (CNS); and exon 1δ was expressed at relatively high levels in the developing CNS. Furthermore, in silico analysis of common putative transcription factor binding sites in the upstream regions of the first exons of both human and mouse PDGFRA/Pdgfra genes predicted common (such as Sry, Mzf1, and Cdx) and unique (such as Sox5, Lmo2, and GATA) transcription factors. Our findings show the diversity of the transcriptional regulation of the PDGFRA/Pdgfra gene.

Hemangioblast, including primitive hematopoietic progenitor cells, play an important role in hematopoietic development, however, the underlying mechanism for the propagation of hematopoietic progenitor cells remains elusive. A variety of regulatory molecules activated in early embryonic development play a critical role in the maintenance of function of hematopoietic progenitor cells. Homeobox transcription factors are an important class of early embryonic developmental regulators determining hematopoietic development. However, the effect of homeobox protein Hox-B4 (HOXB4) ectopic expression on the development of hemangioblasts has not been fully addressed. This study aimed to investigate the role of Hoxb4a, an ortholog gene of HOXB4 in zebrafish, in the hematopoietic development in zebrafish. A transgenic zebrafish line was established with Cre-loxP system that stably overexpressed enhanced green fluorescent protein (EGFP)-tagged Hoxb4a protein under the control of hemangioblast-specific lmo2 promoter. Overexpression of Hoxb4a in the development of hemangioblasts resulted in a considerable increase in the number of stem cell leukemia (scl) and lmo2-positive primitive hematopoietic progenitor cells occurring in the posterior intermediate cell mass (ICM). Interestingly, Hoxb4a overexpression also disrupted the development of myelomonocytes in the anterior yolk sac and the posterior ICM, without affecting erythropoiesis in the posterior ICM. Taken together, these results indicate that Hoxb4a favours the development of hematopoietic progenitor cells originated from hemangioblasts in vivo.

Enhancers are regulatory elements in genomic DNA that contain specific sequence motifs that are bound by DNA-binding transcription factors. The activity of enhancers is tightly regulated in an integrated and combinatorial manner, thus yielding complex patterns of transcription in different tissues. Identifying enhancers is crucial to understanding the physiological and pathogenic roles of their target genes. The RUNX1 intronic enhancer, eR1, acts in cis to regulate RUNX1 gene expression in hematopoietic stem cells (HSCs) and hemogenic endothelial cells. RUNX1 and other hematopoietic transcription factors TAL1/SCL, GATA2, PU.1, LMO2 and LDB1 bind at this region. Interestingly, recent studies have revealed that this region is involved in a large cluster of enhancers termed a super-enhancer. The RUNX1 super-enhancer is observed in normal HSCs and T-cell acute lymphoblastic leukemia cells. In this review, we describe the discovery of eR1 and its roles in normal development and leukemogenesis, as well as its potential applications in stem cell research.

MicroRNA (MIR) signatures are critical to pathobiology and prognosis of acute myeloid leukemia (AML). MIR223 is expressed at low levels in progenitor cells, whereas high expression is induced by granulocytic differentiation. Novel-targeted therapies through epigenetic manipulation of MIR223 regulators are being explored in AML but correlative data between established clinical prognostic markers and MIR223 expression in AML is lacking. MIR223 has inverse relationship with LMO2 protein expression and our group has recently reported a close association between LMO2 protein expression and chromosomal findings in AML patients. In this study, we examined the expression of MIR223 in a large cohort of AML patients and correlated it with LMO2 protein expression, cytogenetic data, degree of differentiation [French-American and British (FAB)/World Health Organization classifications], and overall survival. MIR223 expression was upregulated in only a subset of patients (37%). Suppression of MIR223 was more frequent among patients with aneuploid karyotype compared with diploid karyotype (P=0.005). In AML, not otherwise specified category, AML with maturation (FAB-M2) showed higher levels of MIR223 when compared with either AML without maturation (FAB M0/M1) (P=0.001); AML with monoblastic differentiation (FAB M4/M5) (P=0.004) or AML with myelodysplasia-related changes (P=0.011). Among cytogenetic risk groups, suppression of MIR223 was universal (>95%) in high-risk group when compared with intermediate-risk group (P=0.004). No correlation between MIR223 and LMO2 protein expression was identified. In conclusion, we have shown that suppression of MIR223 expression, as compared with controls, is associated with lack of differentiation and adverse cytogenetic profile, but unrelated with LMO2 protein expression or overall survival.

BACKGROUND

Gene therapy has been in use to cure hereditary and acquired diseases by incorporating the desired gene into the cells with the help of gammaretroviral vectors. Despite the success of this therapy in X-linked severe combined immunodeficiency syndrome, few patients developed leukemia as a major adverse event due to retroviral insertional mutagenesis within stem cells. In experimental animals also, retroviral-mediated gene transfer technique resulted in the development of leukemia. On the other hand, evidence suggests that mature T cells (TC) are relatively resistant to transformation even after retroviral-mediated transfer of potent oncogenes Tcl1, ΔTrkA and LMO2 with no reported side effects yet.

OBJECTIVE

To further address the safety issue for TC use in gene therapy, this study investigated susceptibility of mature polyclonal TC to malignant transformation by the retroviral-mediated transfer of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogene.

METHODS

Wild-type mature TC, isolated from C57BL/6 donor mice (genetic background Ly5.1) were transduced with gamma-retroviral vectors encoding the potent TC oncogene NPM-ALK or the control vector enhanced green fluorescent protein eGFP. The cells were then transplanted into RAG-1 deficient recipient mice (genetic background Ly5.2).

RESULTS

Two out of five mice from NPM-ALK oncogene group developed leukemia/lymphoma after latency periods (153 and 250 days, respectively). None of the mice from the control group developed any malignancy throughout the observational period.

CONCLUSIONS

Mature polyclonal TC are relatively susceptible to malignant transformation after gamma-retroviral mediated transfer of NPM-ALK oncogene; hence safety of TC use in gene therapy should be further investigated to avoid the possible side-effect of development of leukemia/lymphoma.

This study was aimed to investigate the expression level and mechanism of microRNA-223 and LMO2 in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) cells and the mechanism. MicroRNA-223 mimics was transfected to increase the expression of MicroRNA-223 in the lymphocytes sorted by ficoll separation from the bone marrow mononuclear cells (BMMNC) of ALL and CLL patients. MicroRNA-223 inhibitor was transfected to decrease the expression of the MicroRNA-223 in the lymphocytes of normal controls. Then the expression of the MicroRNA-223 and LMO2 in transfected lymphocytes before and after cultivating for 72 hours were detected by RT-PCR, the apoptosis and cell cycle of these cells were measured by flow cytometery. The results indicated that before the transfection, the expression of MicroRNA-223 in ALL and CLL cells was (433.11 ± 144.88), which was significantly lower than that in the normal lymphocyte (949.59 ± 267.39); the expression of LMO2 was (807.10 ± 238.41), which was significantly higher than that in the normal lymphocytes (455.32 ± 176.83) (P < 0.05); after the transfection, the expression of MicroRNA-223 was (571.86 ± 142.00) in ALL and CLL cells, which was significantly higher than that before transfection (P < 0.05), but the expression of LMO2 was significantly lower than that before transfection (651.97 ± 230.12) (P < 0.05); in the normal control the expression of MicroRNA-223 obviously decreased (646.32 ± 172.93) (P < 0.05), the expression of LMO2 was significantly increased (541.27 ± 158.86.2) (P < 0.05). After transfection, the cell cycle G1/G2 phase and apoptosis changed in ALL and CLL cells. Before transfection the cell ratio in cell cycle G1/G2 phase was (94.75 ± 3.15)%, the cell ratio in S phase was (5.14 ± 3.12)%; after transfection the cell ratio in cell cycle G1/G2 phase was (97.03 ± 2.08)% and obviously increased (P < 0.05), the cell ratio in S phase was (2.97 ± 2.08)% and significantly decreased (P < 0.05). Before transfection the apoptosis rate was (54.47 ± 8.72)%, and obviously was higher than that after transfection (60.48 ± 8.81)%. And in the normal control, the cell ratio in G1/G2 phase was significantly higher than that after transfection [(96.73 ± 2.26)%, (94.55 ± 2.77)%, P < 0.05)], and the cell ratio in S phase was significantly increased [(3.25 ± 2.26)%, (5.45 ± 2.77)% (P < 0.05)]. The apoptotic rate in the ALL and CLL patients was significantly higher than that after the transfection [(54.47 ± 8.72)% vs (60.48 ± 8.81)%, respectively (P < 0.05)]. The apoptotic rate in the normal control was significantly lower than that after the transfection [(59.02 ± 10.20)%, (51.96 ± 10.20)%, respectively (P < 0.05)]. It is concluded that the expression of MicroRNA-223 decreases, and the expression of LMO2 increases in lymphocytic leukemia cells which leads to the lymphocytes over-proliferation and abnormal apoptosis, thus may be one of pathogenesis in lymphocytic leukemia.

For X-linked severe combined immunodeficiency (SCID-X1), the practice of gene therapy has revealed an unusual effect: insertional mutagenesis that can lead to leukemia. Even though incorporation of the retrovirus close to the oncogene for T-cell acute lymphoblastic leukemia (T-ALL), LIM-only protein 2 (LMO2) is observed frequently, but it is not clear why LMO2 expression is affected. It was demonstrated that in all the typical T-ALL oncogenes, there is mainly transcription of LMO2 in CD34+ progenitor cells. LYL1, TAN1, and TAL1 are very important intensification factors that are classically used in the gene therapy for copying LMO2 when they are stimulated. For this reason, oncogenes are susceptible to amalgamation with viruses. The IL-2R-gamma (IL-2 receptor γ chain) was found to be a supporting oncogene to LMO2. Nevertheless it was illustrated that excessive expression of IL-2R-gamma did not affect T-cell growth. In comparison to it, the excessive expression of LMO2 in CD34+ cells can cause ongoing increases in the development of T cells. Conversely, there is no effect on the development of B cells and myeloid cells. This information helps explain why LMO2 is mostly affected by various identified T-ALL oncogenes. In addition, throughout the process of T-cell development, expression of IL2R-gamma mediated by retrovirus may not always be oncogenic. As an alternative, replacement of signals of common IL-7 receptors may increase development of T cells wherever LMO2 was expressed and caused abnormal thymocyte development.

Four out of 10 patients of X-linked severe combined immunodeficiency (X-SCID) were finally developed leukemia after receiving the treatment of gene therapy delivered by gamma-retroviral vectors. This is due to the vector integrated to the proximity of lmo2 etc proto-oncogene promoters, leading to the activation of onco-gene expression, which raises the concern of the bio-safety of gene therapy vectors. Lentiviral vectors, especially self-inactivating lentiviral vectors, are considered to be much safer than gamma-retroviral vectors. However self-inactivating lentiviral vectors also have encountered with some unsafe factors and one of them is the problem of transcriptional "read-through" . During the past years, achievements have been made to reduce lentiviral vector transcriptional read-through, which are reviewed herein.

It is critical to specify a signal that directly drives the transition that occurs between cell states. However, such inferences are often confounded by indirect intercellular communications or secondary transcriptomic changes due to primary transcription factors. Although FGF is known for its importance during mesoderm-to-endothelium differentiation, its specific role and signaling mechanisms are still unclear due to the confounding factors referenced above. Here, we attempted to minimize the secondary artifacts by manipulating FGF and its downstream mediators with a short incubation time before sampling and protein-synthesis blockage in a low-density angioblastic/endothelial differentiation system. In less than 8 h, FGF started the conversion of KDR^low/PDGFRA^low nascent mesoderm into KDR^high/PDGFRA^low angioblasts, and the priming by FGF was necessary to endow endothelial formation 72 h later. Further, the angioblastic conversion was mediated by the FGFR1/BRAF/MEK/ERK pathway in mesodermal cells. Finally, two transcription factors, ETV2 and LMO2, were the early direct functional responders downstream of the FGF pathway, and ETV2 alone was enough to complement the absence of FGF. FGF's selective role in mediating the first-step, angioblastic conversion from mesoderm-to-endothelium thus allows for refined control over acquiring and manipulating angioblasts. The noise-minimized differentiation/analysis platform presented here is well-suited for studies on the signaling switches of other mesodermal-lineage fates as well.

Keywords: Angioblasts; ETV2; Endothelium; FGF; FGFR1/BRAF/MEK/ERK; LMO2; Low-density and defined differentiation system; Mesoderm.

Emerging evidence indicates that thymocyte self-renewal induced by progenitor deprivation carries an oncogenic risk that is modulated by intra-thymic competition from differentiation-committed cells. Here we discuss formative studies demonstrating that, in mice, early thymocytes acquire self-renewing potential when thymic progenitor supply is sub-physiological and the importance of cellular competition with this at-risk cell population to prevent lymphoid malignancy. We also consider the possibility that increased thymic residency time, established under conditions of limited cellular competition, may have contributed to oncogenesis observed in early SCID-X1 trials when combined with insertional activation of proto-oncogenes such as LMO2.

OBJECTIVE

The LMO2 gene encodes a transcription factor that regulates key events in erythropoiesis, angiogenesis, and embryogenesis and is highly expressed at the most immature stages of lymphopoiesis. Its implication in Hodgkin lymphoma (HL), Burkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL) is limited in the literature.

METHODS

An immunohistochemical study was performed on 68 lymphoma specimens showing different types including Hodgkin lymphoma (23), Burkitt lymphoma (10) and diffuse large B cell lymphoma (35). Also, seven specimens of the reactive nodal tissue were included as control. A monoclonal anti-human antibody has been used to detect LMO2.

RESULTS

LMO2 was detected in all cases of HL (100%), in nine cases of BL (90%) and in all cases of DLBCL of germinal center (GC) subtype 20/35 (57.1%) but is completely negative in non-germinal center (NGC) DLBCL. In normal control of reactive nodes, LMO2 was expressed in germinal center area but not expressed in other areas including mantle, marginal, or T cell zones. In DLBCL; there was no statistically significant relation between LMO2 positive cases and the studied clinicopathological parameters including patient's age, sex and tumor site, stage and histological subtype. On the other hand, it was statistically significant regarding immunophenotyping of GC versus NGC.

CONCLUSIONS

LMO2 expression is a special feature of GC DLBCL which can be used as a diagnostic marker and therapeutic target. Further studies regarding its prognostic role in patients are recommended.

BACKGROUND LMO2 belongs to the LIM-Only group of LIM domain protein superfamily. It is ubiquitously expressed in different types of tissues and locates either in the nucleus or in the cytoplasm depending on the tissue type. Till now the unique function of LMO2 was considered to be serving as a bridging or blocking molecule that mediates extensive protein-protein interactions. However, the exactly biological features of LMO2 interactome as well as LMO2 function spectrum remain largely unclear. MATERIAL AND METHODS In this study, yeast 2-hybrid assay was firstly performed using LMO2 as the bait and the characteristic of LMO2 protein interactome was analyzed according to the yeast 2-hybrid data and other relative biological information primarily using bioinformatic method. RESULTS Our data indicated that LMO2 favored interacting with peptides containing ß-sheet structure and having relatively unstable confirmation. Moreover, several LMO2 favored interacting domains were identified, including WD40 repeat, coiled-coil, Ankyrin repeat, Zinc finger, PDZ, and SH3, and functions of these domain-containing members were dramatically enriched in some types of cancers. CONCLUSIONS Our results revealed a LMO2 favored protein-interaction pattern in both secondary structure and domain level, and concentrated LMO2 function in kinds of cytoplasmic metabolism pathways as well as multiple types of cancers.

Background: PCNSL is a rare extranodal NHL with poor prognosis. Tumorigenesis has been associated with hyperactivation of BCR downstream and NFkB pathways. We studied the prognosis of the relative expression profile of target genes of NFkB pathway (MYC, BCL2), the essential transcriptional regulator in hematopoiesis LMO2, the checkpoint regulation pathway MGMT, the transcription factor POU2F1, the immune checkpoint gene PDCD1, and the proto-oncogene and transcriptional repressor gene BCL6 and its proteins in PCNSL.

Methods: This study is a retrospective cohort study; 35 immunocompetent PCNSL-DLBCL patients had their gene expression (RT-qPCR) normalized to internal control gene GUSB.

Results: Median patient age was 62 years, median OS was 42.6 months (95% CI: 26.6-58.6), PFS was 41 months (95% CI: 19.7-62.4), and DFS was 59.2 months (95% CI 31.9-86.6). A moderate correlation was found between the gene/protein expressions of MYC (kappa = 0.596, p = .022) and of BCL2 (kappa = 0.426, p = .042). Relative gene expression of MYC ≥ 0.201 (HR 6.117; p = .003) was associated with worse 5-year OS. Relative gene expression of MYC ≥ 0.201 (HR 3.96; p = .016) and MGMT ≥ 0.335 (HR 3.749; p = .056) was associated with worse PFS. Age > 60 years and IELSG score moderate/high were also associated with worse prognosis.

Conclusions: Overexpression of MYC and overexpression of MGMT were prognostic markers associated with unfavorable clinical outcomes in PCNSL.

Keywords: biomarkers; central nervous system neoplasms; gene expression; lymphoma; survival analysis.

LMO2 is a bridging factor within a DNA binding complex and is required for definitive haematopoiesis to occur. The developmental stage of the block in haematopoietic specification is not known. We show that Lmo2-/- mouse embryonic stem cells differentiated to Flk-1+ haemangioblasts, but less efficiently to haemogenic endothelium, which only produced primitive haematopoietic progenitors. Genome-wide approaches indicated that LMO2 is required at the haemangioblast stage to position the TAL1/LMO2/LDB1 complex to regulatory elements that are important for the establishment of the haematopoietic developmental program. In the absence of LMO2, the target site recognition of TAL1 is impaired. The lack of LMO2 resulted in altered gene expression levels already at the haemangioblast stage, with transcription factor genes accounting for ∼15% of affected genes. Comparison of Lmo2-/- with Tal1-/- Flk-1+ cells further showed that TAL1 was required to initiate or sustain Lmo2 expression.

GATA1 is an essential transcriptional regulator of myeloid hematopoietic differentiation towards red blood cells. During erythroid differentiation, GATA1 forms different complexes with other transcription factors such as LDB1, TAL1, E2A and LMO2 'he LDB1 complex') or with FOG1. The functions of GATA1 complexes have been studied extensively in definitive erythroid differentiation; however, the temporal and spatial formation of these complexes during erythroid development is unknown. We applied proximity ligation assay (PLA) to detect, localize and quantify individual interactions during ES cell differentiation and in mouse fetal liver tissue. We show that GATA1/LDB1 interactions appear before the proerythroblast stage and increases in a subset of the CD71+/TER119- cells to activate the terminal erythroid differentiation program in 12.5 day fetal liver. Using Ldb1 and Gata1 knockdown fetal liver cells, we studied the functional contribution of the GATA1/LDB1 complex during differentiation. This shows that the active LDB1 complex appears quite late at the proerythroblast stage of differentiation and confirms the power of PLA in studying the dynamic interaction of proteins in cell differentiation at the single cell level. We provide dynamic insight in the temporal and spatial formation of the GATA1 and LDB1 transcription factor complexes during hematopoietic development and differentiation.

Mechanisms of stromal-epithelial crosstalk are essential for Prostate cancer (PCa) tumorigenesis and progression. Peripheral zone of the prostate gland possesses a stronger inclination for PCa than transition zone. We previously found a variety of genes that differently expressed among different prostate stromal cells, including LIM domain only 2 (LMO2) which highly expressed in peripheral zone derived stromal cells (PZSCs) and PCa associated fibroblasts (CAFs) compared to transition zone derived stromal cells (TZSCs). Studies on its role in tumors have highlighted LMO2 as an oncogene. Herein, we aim to study the potential mechanisms of stromal LMO2 in promoting PCa progression. The in vitro cells co-culture and in vivo cells recombination revealed that LMO2 over-expressed prostate stromal cells could promote the proliferation and invasiveness of either prostate epithelial or cancer cells. Further protein array screening confirmed that stromal LMO2 stimulated the secretion of Interleukin-11 (IL-11), which could promote proliferation and invasiveness of PCa cells via IL-11 receptor α (IL11Rα) - STAT3 signaling. Moreover, stromal LMO2 over-expression could suppress miR-204-5p which was proven to be a negative regulator of IL-11 expression. Taken together, results of our study demonstrate that prostate stromal LMO2 is capable of stimulating IL-11 secretion and by which activates IL11Rα - STAT3 signaling in PCa cells and then facilitates PCa progression. These results may make stromal LMO2 responsible for zonal characteristic of PCa and as a target for PCa microenvironment-targeted therapy.

OBJECTIVE

To investigate function of the Lim-only protein(LMO2) in hemangioblast generated from murine embryonic stem cells differentiation to hematopoietic cells.

METHODS

The hemangioblast-specific expression vector with lmo2 or green fluorescence protein gene was constructed, respectively. The murine embryonic stem cells were transfected by the hemangioblast-specific expression vectors. The neomycin-resistance ES cell clones were obtained after having been screened by G418. The cell clones were spontaneously differentiated into embryo bodies(EB) containing hemangioblast.Expression of the hematopoietic genes was investigated by real-time reverse transcription-ploymerase chain reaction during EB differentiation.For the EB cells, blast-cloning forming cells analysis and blood-colony forming unit analysis were then performed, respectively. The numbers of the blasts were counted during hematopoietic differentiation.

RESULTS

The hemangioblast-specific expression vector with lmo2 or green fluorescence protein was transfected into ES cells.The neomycin-resistance ES cells generated EBs from 2.5 days to 10 days.Real time reverse transcription-ploymerase chain reaction analysis indicated that overexpression of lmo2 increased the expression of hematopoietic genes(gata1, tal1, Β-h1, and Β-major globin) during EB formation.Blast-cloning forming cells analysis showed that the numbers of the blasts generated by ES/lmo2 was 2-or 3-fold than those in the controls.The total numbers of the blood-colony forming unit or the numbers of the erythrocyte colony-forming unit generated by ES/lmo2 were 2.5 times or 3 times, respectively, when compared with the controls.

CONCLUSIONS

LMO2 enhances the proliferation and differentiation of hemangioblasts.

The development of macrophages is a highly regulated process requiring coordination amongst transcription factors. The presence/absence, relative levels, antagonism, or synergy of all transcription factors involved is critical to directing lineage cell fate and differentiation. While relative levels of many key myeloid transcription factors have been determined in mammalian macrophage differentiation, a similar set of studies have yet to be conducted in a teleost system. In this study, we report on the mRNA levels of transcription factors (cebpa, cjun, cmyb, egr1, gata1, gata2, gata3, lmo2, mafb, pax5, pu.1 and runx1) in sorted goldfish progenitor cells, monocytes, and macrophages from primary kidney macrophage cultures. The mRNA levels of runx1 and pu.1 were significantly higher, gata3 and pax5 mRNA levels were lower, in monocytes compared to progenitors, and the mRNA levels of cjun, egr1, gata2, gata3, mafb and pax5 were significantly decreased in macrophages compared to progenitor cells. The relative mRNA levels of the interferon regulatory factor family of transcription factors, irf1, irf2, irf5, irf7, irf8 and irf9 in sorted progenitors, monocytes and macrophages were also measured. In contrast to other irf family transcription factors examined, irf8 mRNA levels were increased in monocytes compared to progenitors by greater than three-fold, suggesting that irf8 is important for monopoiesis. Lastly, we show the differential regulation of myeloid transcription factor mRNA levels in sorted progenitor cells from 1, 2, or 3-day old cultures in response to the recombinant goldfish growth factors, rgCSF-1 and rgKITLA.

Haematopoiesis is a complex process in which the regulatory mechanisms of several implicated transcription factors remain uncertain. Drosophila melanogaster is an excellent model to resolve the unanswered questions about the blood cell development. This study describes the role of Beadex, a Drosophila homologue of LIM domain only 2 (LMO2), in haematopoiesis. Mutants of Beadex were analysed for blood cell abnormalities. Crystal cells, a subset of haemocytes, were significantly more in Beadex hypermorphic flies. Similarly, Beadex misexpression in prohemocytes altered the crystal cell numbers. Stage-specific misexpression analyses demonstrated that Beadex functions after the prohemocytes enter the crystal cell lineage. We also discovered that Pannier-U-shaped complex is a negative regulator of the crystal cell differentiation and is possibly negatively regulated by Beadex through its interaction with Pannier. We, therefore, suggest the mechanism of two novel regulators of crystal cell specification-Beadex and Pannier-during Drosophila haematopoiesis.