Embryonic-Derived Myb- Macrophages Enhance Bacterial Clearance and Improve Survival in Rat Sepsis.

Peritoneal resident macrophages play a key role in combating sepsis in the peritoneal cavity. We sought to determine if peritoneal transplantation of embryonic Myb- "peritoneal-like" macrophages attenuate abdominal fecal sepsis. Directed differentiation of rodent pluripotent stem cells (PSCs) was used in factor-defined media to produce embryonic-derived large "peritoneal-like" macrophages (Ed-LPM) that expressed peritoneal macrophage markers and demonstrated phagocytic capacity. Preclinical in vivo studies determined Ed-LPM efficacy in rodent abdominal fecal sepsis with or without Meropenem. Ex vivo studies explored the mechanism and effects of Ed-LPM on host immune cell number and function, including phagocytosis, reactive oxygen species (ROS) production, efferocytosis and apoptosis. Ed-LPM reduced sepsis severity by decreasing bacterial load in the liver, spleen and lungs. Ed-LPM therapy significantly improved animal survival by ~30% and reduced systemic bacterial burden to levels comparable to Meropenem therapy. Ed-LPM therapy decreased peritoneal TNFα while increasing IL-10 concentrations. Ed-LPMs enhanced peritoneal macrophage phagocytosis of bacteria, increased macrophage production of ROS and restored homeostasis via apoptosis and efferocytosis-induced clearance of neutrophils. In conclusion, Ed-LPM reduced systemic sepsis severity, improved survival and reduced bacterial load by enhancing peritoneal macrophage bacterial phagocytosis and killing and clearance of intra-peritoneal neutrophils. Macrophage therapy may be a potential strategy to address sepsis.

Yolk sac, but not hematopoietic stem cell-derived progenitors, sustain erythropoiesis throughout murine embryonic life.

In the embryo, the first hematopoietic cells derive from the yolk sac and are thought to be rapidly replaced by the progeny of hematopoietic stem cells. We used three lineage-tracing mouse models to show that, contrary to what was previously assumed, hematopoietic stem cells do not contribute significantly to erythrocyte production up until birth. Lineage tracing of yolk sac erythromyeloid progenitors, which generate tissue resident macrophages, identified highly proliferative erythroid progenitors that rapidly differentiate after intra-embryonic injection, persisting as the major contributors to the embryonic erythroid compartment. We show that erythrocyte progenitors of yolk sac origin require 10-fold lower concentrations of erythropoietin than their hematopoietic stem cell-derived counterparts for efficient erythrocyte production. We propose that, in a low erythropoietin environment in the fetal liver, yolk sac-derived erythrocyte progenitors efficiently outcompete hematopoietic stem cell progeny, which fails to generate megakaryocyte and erythrocyte progenitors.

Sorghum 3-Deoxyanthocyanidin Flavonoids Confer Resistance against Corn Leaf Aphid.

In this study we examined the role of sorghum flavonoids in providing resistance against corn leaf aphid (CLA) Rhopalosiphum maidis. In sorghum, accumulation of these flavonoids is regulated by a MYB transcription factor, yellow seed1 (y1). Functional y1 alleles accumulate 3-deoxyflavonoids (3-DFs) and 3-deoxyanthocyanidins (3-DAs) whereas null y1 alleles fail to accumulate these compounds. We found that significantly higher numbers of alate CLA adults colonized null y1 plants as compared to functional y1 plants. Controlled cage experiments and pairwise choice assays demonstrated that apterous aphids preferred to feed and reproduce on null y1 plants. These near-isogenic sorghum lines do not differ in their epicuticular wax content and were also devoid of any leaf trichomes. Significantly higher mortality of CLA was observed on artificial aphid diet supplemented with flavonoids obtained from functional y1 plants as compared to null y1 plants or the relevant controls. Our results demonstrate that the proximate mechanism underlying the deleterious effects on aphids is y1-regulated flavonoids which are important defense compounds against CLA.

c-Myb Regulates the T-Bet-Dependent Differentiation Program in B Cells to Coordinate Antibody Responses.

Humoral immune responses are tailored to the invading pathogen through regulation of key transcription factors and their networks. This is critical to establishing effective antibody-mediated responses, yet it is unknown how B cells integrate pathogen-induced signals to drive or suppress transcriptional programs specialized for each class of pathogen. Here, we detail the key role of the transcription factor c-Myb in regulating the T-bet-mediated anti-viral program. Deletion of c-Myb in mature B cells significantly increased serum IgG2c and CXCR3 expression by upregulating T-bet, normally suppressed during Th2-cell-mediated responses. Enhanced expression of T-bet resulted in aberrant plasma cell differentiation within the germinal center, mediated by CXCR3 expression. These findings identify a dual role for c-Myb in limiting inappropriate effector responses while coordinating plasma cell differentiation with germinal center egress. Identifying such intrinsic regulators of specialized antibody responses can assist in vaccine design and therapeutic intervention in B-cell-mediated immune disorders.

An ancient transcription factor initiates the burst of piRNA production during early meiosis in mouse testes.

Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during postnatal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feedforward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals.

c-Myb and GATA-1 alternate dominant roles during megakaryocyte differentiation.

BACKGROUND: Transcription factors are essential for blood cell formation. Mice expressing low levels of c-Myb (c-Myb(low)) have an increased number of bone marrow megakaryocytes (MKs) and corresponding thrombocytosis. In contrast, mice engineered to express low levels of GATA-1 (GATA-1(low)) in the megakaryocytic lineage exhibit aberrant megakaryocytopoiesis with hyperproliferation of progenitors and defective terminal differentiation leading to thrombocytopenia. These seemingly opposite roles may affect platelet turnover and thus be of clinical relevance. OBJECTIVE: To determine how these two transcription factors act together to control megakaryocytopoiesis and platelet formation. METHODS: We used a combination of cellular and molecular in vitro assays to examine the ability of bone marrow cells from mice expressing low levels of both c-Myb and GATA-1 (referred to as double(low)) to produce MKs and platelets. RESULTS: Double(low) cells, or those with low GATA-1 levels in which c-Myb is conditionally deleted, lack the hyperproliferative capacity of GATA-1(low) cells, allowing the cells to proceed towards more committed MKs that are, however, impaired in their capacity to produce fully differentiated cells, as confirmed by the abundance of morphologically aberrant cells that lack the ability to form proplatelets. CONCLUSION: c-Myb and GATA-1 act in concert to achieve correct megakaryocytic differentiation. GATA-1 regulates both the proliferation of megakaryocytic progenitors and their terminal maturation. c-Myb also acts at the level of the progenitor by influencing its commitment to differentiation, but in contrast to GATA-1 it does not have any effect on the process of terminal differentiation.

c-Myb promotes the survival of CD4+CD8+ double-positive thymocytes through upregulation of Bcl-xL.

Mechanisms that regulate the lifespan of CD4(+)CD8(+) double-positive (DP) thymocytes help shape the peripheral T cell repertoire. However, the molecular mechanisms controlling DP thymocyte survival remain poorly understood. The Myb proto-oncogene encodes a transcription factor required during multiple stages of T cell development. We demonstrate that Myb mRNA expression is upregulated as thymocytes differentiate from the double-negative into the metabolically quiescent, small, preselection DP stage during T cell development. Using a conditional deletion mouse model, we demonstrate that Myb-deficient DP thymocytes undergo premature apoptosis, resulting in a limited Tcralpha repertoire biased toward 5' Jalpha segment usage. Premature apoptosis occurs specifically in the small preselection DP compartment in an alphabetaTCR-independent manner and is a consequence of decreased Bcl-xL expression. Forced Bcl-xL expression is able to rescue survival, and reintroduction of c-Myb restores both Bcl-xL expression and the small preselection DP compartment. We further demonstrate that c-Myb promotes transcription at the Bcl2l1 locus via a genetic pathway that is independent of the expression of T cell-specific factor-1 or RORgammat, two transcription factors that induce Bcl-xL expression in T cell development. Thus, Bcl-xL is a novel mediator of c-Myb activity during normal T cell development.

c-Myb is required for pro-B cell differentiation.

The c-Myb transcription factor is required for normal adult hematopoiesis. However, the embryonic lethality of Myb-null mutations has been an impediment to identifying roles for c-Myb during lymphocyte development. We have used tissue-specific inactivation of the Myb locus in early progenitor cells to demonstrate that c-Myb is absolutely required for the differentiation of CD19(+) B-lineage cells and B cell differentiation is profoundly blocked beyond the pre-pro-B cell stage in Myb(f/f) Mb1-cre mice. We demonstrate that c-Myb is required for the intrinsic survival of CD19(+) pro-B cells as well as the proper expression of the alpha-chain of the IL-7 receptor (CD127) and Ebf1. However, survival of c-Myb-deficient CD19(+) pro-B cells cannot be rescued by transduction with CD127-producing retrovirus, suggesting that c-Myb controls a survival pathway independent of CD127. Furthermore, c-Myb-deficient progenitor cells inefficiently generate CD19(+) B-lineage cells during stromal cell culture but this process can be partially rescued with exogenous Ebf1. Thus, c-Myb does not appear to be required for commitment to B cell differentiation but is crucial for B cell differentiation to the CD19(+) pro-B cell stage as well as survival of CD19(+) pro-B cells. Surprisingly, forced c-Myb expression in lymphoid-primed multipotent progenitors favors differentiation toward the myeloid lineage, suggesting that proper c-Myb expression is crucial for B-lineage development.

Primitive erythropoiesis and megakaryopoiesis in the yolk sac are independent of c-myb.

Hematopoiesis initiates within the yolk sac of mammalian embryos in overlapping primitive and definitive waves, each containing erythroid and megakaryocyte progenitors. c-myb-null mouse fetuses lack definitive erythrocytes but contain primitive erythroblasts and hepatic megakaryocytes. However, it is unclear if c-myb-null embryos harbor definitive erythroid or any megakaryocyte progenitors. We determined that c-myb was not expressed in primitive erythroid precursors and that c-myb-null embryos had normal primitive erythroid and megakaryocyte progenitor numbers and kinetics between embryonic day (E) 7.0 and E9.0. While primitive hematopoiesis is c-myb-independent, no definitive erythroid potential was detected in c-myb-null embryos, confirming that definitive erythropoiesis, beginning at E8.25 in the yolk sac, is completely c-myb-dependent. In contrast, reduced numbers of megakaryocyte progenitors with restricted proliferative capacity persist in E10.5 yolk sac and E11.5 liver. Despite this impaired megakaryocyte potential, c-myb-null fetuses had normal platelet numbers at E12.5 but became thrombocytopenic by E15.5, suggesting that c-myb is required for sustained thrombopoiesis.

Loss of Drosophila Myb interrupts the progression of chromosome condensation.

Completion of chromosome condensation is required before segregation during the mitotic cell cycle to ensure the transmission of genetic material with high fidelity in a timely fashion. In many eukaryotes this condensation is regulated by phosphorylation of histone H3 on Ser 10 (H3S10). This phosphorylation normally begins in the late-replicating pericentric heterochromatin and then spreads to the earlier replicating euchromatin. Here, we show that these phases of condensation are genetically separable in that the absence of Drosophila Myb causes cells to arrest with H3S10 phosphorylation of heterochromatin but not euchromatin. In addition, we used mosaic analysis to demonstrate that although the Myb protein can be removed in a single cell cycle, the failure of chromosome condensation occurs only after many cell divisions in the absence of Myb protein. The Myb protein is normally located in euchromatic but not heterochromatic regions of the nucleus, suggesting that Myb may be essential for a modification of euchromatin that is required for the efficient spread of chromosome condensation.

Coordination of erythropoiesis by the transcription factor c-Myb.

The involvement of the transcription factor c-Myb in promoting the proliferation and inhibition of erythroid cell differentiation has been established in leukemia cell models. The anemia phenotype observed in c-myb knockout and knockdown mice highlights a critical role for c-Myb in erythropoiesis. However, determining the reason for the failure of erythropoiesis in these mice and the precise function of c-Myb in erythroid progenitors remains elusive. We examined erythroid development under conditions of reduced c-Myb protein levels and report an unexpected role for c-Myb in the promotion of commitment to the erythroid lineage and progression to erythroblast stages. c-myb knockdown erythroid colony-forming unit (CFU-E) stage progenitors displayed an immature phenotype and aberrant expression of several hematopoietic regulators. To extend our findings, we analyzed the response of normal enriched erythroid progenitors to inducible disruption of a floxed c-myb allele. In agreement with the c-myb knockdown phenotype, we show that c-Myb is strictly required for expression of the c-Kit receptor in erythroid cells.

Disruption of B-myb in DT40 cells reveals novel function for B-Myb in the response to DNA-damage.

B-Myb is a highly conserved vertebrate member of the Myb transcription factor family, which is expressed in virtually all proliferating cells. A large body of evidence suggests that B-Myb plays an important role in cell cycle regulation; however, the exact nature of its function has not yet been clarified. We have used gene targeting in chicken DT40 cells, a cell line exhibiting very high rates of homologous recombination, to create cells expressing endogenous B-myb in a doxycyclin-dependent manner. We find that the cells proliferate well in the absence of B-Myb, suggesting that B-Myb is not essential for cell proliferation per se. However, cells lacking B-Myb are more sensitive to DNA-damage induced by UV-irradiation and alkylation. Our work provides the first direct evidence for a novel function of B-Myb in the response to DNA-damage. The cells described here should be a useful model to characterize this function in more detail.

Requirement of c-myb in T cell development and in mature T cell function.

Previous reports have suggested that the protooncogene c-myb participates in T cell development in the thymus and mature T cell proliferation. We have generated two T cell-specific c-myb knockout mouse models, myb/LckCre and myb/CD4Cre. We have demonstrated that c-myb is required for the development of thymocytes at the DN3 stage, for survival and proliferation of double-positive thymocytes, for differentiation of single-positive CD4 and CD8 T cells, and for the proliferative responses of mature T cells. In addition, our data show that c-myb is directly involved in the formation of double-positive CD4+CD8+CD25+, CD4+CD25+, and CD8+CD25+ T cells, developmental processes that may imply a role for c-myb in autoimmune dysfunction.

c-myb Heterozygous mice are hypersensitive to 5-fluorouracil and ionizing radiation.

Hypersensitivity to chemo- and radiotherapy employed during cancer treatment complicates patient management. Identifying mutations in genes that compromise tissue recovery would rationalize treatment and may spare hypersensitive patients undue tissue damage. Genes that govern stem cell homeostasis, survival, and progenitor cell maintenance are of particular interest in this regard. We used wild-type and c-myb knock-out mice as model systems to explore stem and progenitor cell numbers and sensitivity to cytotoxic damage in two radiosensitive tissue compartments, the bone marrow and colon. Because c-myb null mice are not viable, we used c-myb heterozygous mice to test for defects in stem-progenitor cell pool recovery following gamma-radiation and 5-fluorouracil treatment, showing that c-myb(+/-) mice are hypersensitive to both agents. While apoptosis is comparable in mutant and wild-type mice following radiation exposure, the crypt beds of c-myb(+/-) mice are markedly depleted of proliferating cells. Extrapolating from these data, we speculate that acute responses to cytotoxic damage in some patients may also be attributed to compromised c-myb function.

Critical functions for c-Myb at three checkpoints during thymocyte development.

The transcription factor c-Myb is expressed throughout T cell development in the thymus. However, little is understood about c-Myb function because of the embryonic lethality of traditional Myb-null mutations. Using tissue-specific deletion to abrogate c-Myb expression at distinct stages of T cell development, we identify three points at which c-Myb activity is required for normal T cell differentiation: transition through the double-negative 3 stage, survival of preselection CD4(+)CD8(+) thymocytes, and differentiation of CD4 thymocytes. Thus, c-Myb is essential at several stages during T cell development in the thymus.

Progression through key stages of haemopoiesis is dependent on distinct threshold levels of c-Myb.

The c-Myb transcription factor is expressed in immature haemopoietic cells and at key stages during differentiation. Loss of the c-myb gene results in embryonic lethality because mature blood cells fail to develop, although commitment to definitive haemopoiesis occurs. We have generated a knockdown allele of c-myb, expressing low levels of the protein, which has enabled us to investigate further the involvement of c-Myb in haemopoiesis. Low levels of c-Myb are sufficient to allow progenitor expansion but, importantly, we show that progression of progenitors towards terminal differentiation is significantly altered. Suboptimal levels of c-Myb favour differentiation of macrophage and megakaryocytes, while higher levels seem to be particularly important in the control of erythropoiesis and lymphopoiesis. We provide evidence that the transition from the CFU-E to erythroblasts is critically dependent on c-Myb levels. During thymopoiesis, c-Myb appears to regulate immature cell numbers and differentiation prior to expression of CD4 and CD8. Overall, our results point to a complex involvement of c-Myb in the regulation of proliferation and commitment within the haemopoietic hierarchy.

Targeted disruption of c-myb in the chicken pre B-cell line DT40.

The c-myb proto-oncogene is highly expressed in a wide variety of immature hematopoietic cells and plays a key role in the development of the hematopoietic system. c-myb and its retroviral counterpart v-myb encode transcription factors which have been implicated in the regulation of certain target genes. Targeting of c-myb in mouse embryonic stem cells by homologous recombination has provided clear evidence that c-myb is necessary for the proper development of most myeloid lineages of the hematopoietic system as well as of T-lymphocytes. Here we have explored the function of c-myb in the B-lymphoid lineage. We have used the chicken DT40 cells, a pre B-cell line which shows extremely high efficiencies of homologous recombination, as a model system to disrupt c-myb. DT40 cells lacking a functional c-myb gene are viable and show only minor perturbations of their growth parameters, indicating that c-myb is not an essential gene in these cells. We have used the c-myb null DT40 cells to analyse the expression of genes which have been previously been identified as myb target genes. Neither c-myc nor bcl-2, two putative myb targets, showed altered expression in the cells lacking c-myb. However, expression of the Pdcd4 gene, a myb target gene originally identified in a myelomonocytic cell line expressing a conditional form of v-myb, was diminished in the absence of c-myb. The c-myb knock-out cells described here should provide a useful model system for the identification and characterization of c-myb target genes in B-lymphoid cells.

c-Myb is critical for murine colon development.

The mammalian colon develops from a simple tube of undifferentiated cells into a complex, highly ordered organ, with a continuously self-renewing epithelial layer. We have previously described c-Myb expression in the epithelia of murine and human colon crypts and documented increased expression in colorectal adenocarcinoma cells. To investigate the role of c-Myb in colonic epithelium development, we have used embryos with a disrupted c-myb gene. Prior to the in utero death of these embryos at E15, we excised colon tissue and transplanted it under the kidney capsule of recipient mice to allow further development and cyto-differentiation. Compared to the colons of wildtype and heterozygous littermates, the c-myb homozygous knockout colon is highly irregular with a disordered epithelium and abnormal crypts. In addition, the expression of Bcl-2, a known target of c-Myb, is reduced and apoptosis is increased, indicating a critical requirement for c-Myb in normal colon development.