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Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.

Macrophages play essential roles throughout the wound repair process. Nevertheless, mechanisms regulating the process are poorly understood. MAFB is specifically expressed in the macrophages in hematopoietic tissue and is vital to homeostatic function. Comparison of the skin wound repair rates in macrophage-specific, MAFB-deficient mice (

Focal segmental glomerulosclerosis (FSGS) is a common cause of steroid-resistant nephrotic syndrome. Spontaneous remission of FSGS is rare and steroid-resistant FSGS frequently progresses to renal failure. Many inheritable forms of FSGS have been described, caused by mutations in proteins that are important for podocyte function. Here, we show that a basic leucine zipper transcription factor, MafB, protects against FSGS. MAFB expression was found to be decreased in the podocytes of patients with FSGS. Moreover, conditional podocyte-specific MafB-knockout mice developed FSGS with massive proteinuria accompanied by depletion of the slit diaphragm-related proteins (Nphs1 and Magi2), and the podocyte-specific transcription factor Tcf21. These findings indicate that MafB plays a crucial role in the pathogenesis of FSGS. Consistent with this, adriamycin-induced FSGS and attendant proteinuria were ameliorated by MafB overexpression in the podocytes of MafB podocyte-specific transgenic mice. Thus, MafB could be a new therapeutic target for FSGS.

Non-alcoholic fatty liver disease (NAFLD) constitutes a metabolic disorder with high worldwide prevalence and increasing incidence. The inflammatory progressive state, non-alcoholic steatohepatitis (NASH), leads to liver fibrosis and carcinogenesis. Here, we evaluated whether tyrosinase mutation underlies NASH pathophysiology. Tyrosinase point-mutated B6 (Cg)-Tyrc-2J/J mice (B6 albino) and C57BL/6J black mice (B6 black) were fed with high cholesterol diet (HCD) for 10 weeks. Normal diet-fed mice served as controls. HCD-fed B6 albino exhibited high NASH susceptibility compared to B6 black, a phenotype not previously reported. Liver injury occurred in approximately 50% of B6 albino from one post HCD feeding, with elevated serum alanine aminotransferase and aspartate aminotransferase levels. NASH was induced following 2 weeks in severe-phenotypic B6 albino (sB6), but B6 black exhibited no symptoms, even after 10 weeks. HCD-fed sB6 albino showed significantly higher mortality rate. Histological analysis of the liver revealed significant inflammatory cell and lipid infiltration and severe fibrosis. Serum lipoprotein analysis revealed significantly higher chylomicron and very low-density lipoprotein levels in sB6 albino. Moreover, significantly higher small intestinal lipid absorption and lower fecal lipid excretion occurred together with elevated intestinal NPC1L1 expression. As the tyrosinase point mutation represents the only genetic difference between B6 albino and B6 black, our work will facilitate the identification of susceptible genetic factors for NASH development and expand the understanding of NASH pathophysiology.

The transcription factor MafB is specifically expressed in macrophages. We have recently demonstrated that MafB is expressed in anti-inflammatory alternatively activated M2 macrophages in vitro. Tumor-associated macrophages (TAMs) are a subset of M2 type macrophages that can promote immunosuppressive activity, induce angiogenesis, and promote tumor cell proliferation. To examine whether MafB express in TAMs, we analyzed green fluorescent protein (GFP) expression in Lewis lung carcinoma tumors of MafB-GFP knock-in heterozygous mice. FACS analysis demonstrated GFP fluorescence in cells positive for macrophage-markers (F4/80, CD11b, CD68, and CD204). Moreover, quantitative RT-PCR analysis with F4/80+GFP+ and F4/80+GFP− sorted cells showed that the GFP-positive macrophages express IL-10, Arg-1, and TNF-α, which were known to be expressed in TAMs. These results indicate that MafB is expressed in TAMs. Furthermore, immunostaining analysis using an anti-MAFB antibody revealed that MAFB is expressed in CD204-and CD68-positive macrophages in human lung cancer samples. In conclusion, MafB can be a suitable marker of TAMs in both mouse and human tumor tissues.

Zinc-finger transcription factors GATA2 and GATA3 are both expressed in the developing inner ear, although their overlapping versus distinct activities in adult definitive inner ear are not well understood. We show here that GATA2 and GATA3 are co-expressed in cochlear spiral ganglion cells and redundantly function in the maintenance of spiral ganglion cells and auditory neural circuitry. Notably, Gata2 and Gata3 compound heterozygous mutant mice had a diminished number of spiral ganglion cells due to enhanced apoptosis, which resulted in progressive hearing loss. The decrease in spiral ganglion cellularity was associated with lowered expression of neurotrophin receptor TrkC that is an essential factor for spiral ganglion cell survival. We further show that Gata2 null mutants that additionally bear a Gata2 YAC (yeast artificial chromosome) that counteracts the lethal hematopoietic deficiency due to complete Gata2 loss nonetheless failed to complement the deficiency in neonatal spiral ganglion neurons. Furthermore, cochlea-specific Gata2 deletion mice also had fewer spiral ganglion cells and resultant hearing impairment. These results show that GATA2 and GATA3 redundantly function to maintain spiral ganglion cells and hearing. We propose possible mechanisms underlying hearing loss in human GATA2- or GATA3-related genetic disorders.

MAFB is a basic leucine zipper (bZIP) transcription factor specifically expressed in macrophages. We have previously identified MAFB as a candidate marker for tumor-associated macrophages (TAMs) in human and mouse models. Here, we analyzed single-cell sequencing data of patients with lung adenocarcinoma obtained from the GEO database (GSE131907). Analyzed data showed that general macrophage marker CD68 and macrophage scavenger receptor 1 (CD204) were expressed in TAM and lung tissue macrophage clusters, while transcription factor MAFB was expressed specifically in TAM clusters. Clinical records of 120 patients with lung adenocarcinoma stage I (n = 57), II (n = 21), and III (n = 42) were retrieved from Tsukuba Human Tissue Biobank Center (THB) in the University of Tsukuba Hospital, Japan. Tumor tissues from these patients were extracted and stained with anti-human MAFB antibody, and then MAFB-positive cells relative to the tissue area (MAFB+ cells/tissue area) were morphometrically quantified. Our results indicated that higher numbers of MAFB+ cells significantly correlated to increased local lymph node metastasis (nodal involvement), high recurrence rate, poor pathological stage, increased lymphatic permeation, higher vascular invasion, and pleural infiltration. Moreover, increased amounts of MAFB+ cells were related to poor overall survival and disease-free survival, especially in smokers. These data indicate that MAFB may be a suitable prognostic biomarker for smoker lung cancer patients.

Microgravity induces skeletal muscle atrophy, particularly in the soleus muscle, which is predominantly composed of slow-twitch myofibre (type I) and is sensitive to disuse. Muscle atrophy is commonly known to be associated with increased production of reactive oxygen species. However, the role of NRF2, a master regulator of antioxidative response, in skeletal muscle plasticity during microgravity-induced atrophy, is not known. To investigate the role of NRF2 in skeletal muscle within a microgravity environment, wild-type and Nrf2-knockout (KO) mice were housed in the International Space Station for 31 days. Gene expression and histological analyses demonstrated that, under microgravity conditions, the transition of type I (oxidative) muscle fibres to type IIa (glycolytic) was accelerated in Nrf2-KO mice without affecting skeletal muscle mass. Therefore, our results suggest that NRF2 affects myofibre type transition during space flight., Hayashi et al. report on the effect of space flight on muscle mass, fibre size and type, composition and gene signalling of muscle in WT and Nrf2-KO mice. Their results demonstrate a key role of NRF2 in maintaining the slow-type fibres of soleus muscle during space flight and in microgravity environments.

MAFB is a transcription factor involved in the terminal differentiation of several cell types, including macrophages and keratinocytes. MAFB is also expressed in lymphatic endothelial cells (LECs) and is upregulated by VEGF-C/VEGFR-3 signaling. Recent studies have revealed that MAFB regulates several genes involved in lymphatic differentiation and that global Mafb knockout mice show defects in patterning of lymphatic vessels during embryogenesis. However, it has remained unknown whether this effect is LEC-intrinsic and whether MAFB might also be involved in postnatal lymphangiogenesis. We established conditional, lymphatic-specific Mafb knockout mice and found comparable lymphatic patterning defects during embryogenesis as in the global MAFB knockout. Lymphatic MAFB deficiency resulted in increased lymphatic branching in the diaphragm at P7, but had no major effect on lymphatic patterning or function in healthy adult mice. By contrast, tumor-induced lymphangiogenesis was enhanced in mice lacking lymphatic MAFB. Together, these data reveal that LEC-expressed MAFB is involved in lymphatic vascular morphogenesis during embryonic and postnatal development as well as in pathological conditions. Therefore, MAFB could represent a target for therapeutic modulation of lymphangiogenesis., Angiogenesis, 23 (3)

By using near-infrared fluorescent protein (iRFP)-expressing hematopoietic cells, we established a novel, quantitative, in vivo, noninvasive atherosclerosis imaging system. This murine atherosclerosis imaging approach targets macrophages expressing iRFP in plaques. Low-density lipoprotein receptor-deficient (LDLR−/−) mice transplanted with beta-actin promoter-derived iRFP transgenic (TG) mouse bone marrow (BM) cells (iRFP → LDLR−/−) were used. Atherosclerosis was induced by a nonfluorescent 1.25% cholesterol diet (HCD). Atherosclerosis was compared among the three differently induced mouse groups. iRFP → LDLR−/− mice fed a normal diet (ND) and LDLR−/− mice transplanted with wild-type (WT) BM cells were used as controls. The in vivo imaging system (IVIS) detected an enhanced iRFP signal in the thoracic aorta of HCD-fed iRFP → LDLR−/− mice, whereas iRFP signals were not observed in the control mice. Time-course imaging showed a gradual increase in the signal area, which was correlated with atherosclerotic plaque progression. Oil red O (ORO) staining of aortas and histological analysis of plaques confirmed that the detected signal was strictly emitted from plaque-positive areas of the aorta. Our new murine atherosclerosis imaging system can noninvasively image atherosclerotic plaques in the aorta and generate longitudinal data, validating the ability of the system to monitor lesion progression.

Focal segmental glomerulosclerosis (FSGS) is a leading cause of end-stage renal disease in children and adults. Genetic factors significantly contribute to early-onset FSGS, but the etiologies of most adult cases remain unknown. Genetic studies of monogenic syndromic FSGS exhibiting extra-renal manifestations have uncovered an unexpected biological role for genes in the development of both podocytes and other cellular lineages. To help define these roles, we studied two unrelated families with FSGS associated with Duane Retraction Syndrome, characterized by impaired horizontal eye movement due to cranial nerve malformation. All four affected individuals developed FSGS and Duane Retraction Syndrome in their first to second decade of life, manifested as restricted abduction together with globe retraction and narrowed palpebral fissure on attempted adduction. Hypoplasia of the abducens nerves and hearing impairment occurred in severely affected individuals. Genetic analyses revealed that affected individuals harbor a rare heterozygous substitution (p.Leu239Pro) in MAFB, a leucine zipper transcription factor. Luciferase assays with cultured monocytes indicated that the substitution significantly reduced transactivation of the F4/80 promoter, the known MAFB recognition element. Additionally, immunohistochemistry indicated reduced MAFB expression in the podocytes of patients. Structural modeling suggested that the p.Leu239Pro substitution in the DNA-binding domain possibly interferes with the stability of the adjacent zinc finger. Lastly, podocytes in neonatal mice with p.Leu239Pro displayed impaired differentiation. Thus, MAFB mutations impair development and/or maintenance of podocytes, abducens neurons and the inner ear. The interactions between MAFB and regulatory elements in these developing organs are likely highly specific based on spatiotemporal requirements.

The transcription factor MafB is expressed by monocytes and macrophages. Efferocytosis (apoptotic cell uptake) by macrophages is important for inhibiting the development of autoimmune diseases, and is greatly reduced in Mafb-deficient macrophages. Here, we show the expression of the first protein in the classical complement pathway C1q is important for mediating efferocytosis and is reduced in Mafb-deficient macrophages. The efferocytosis defect in Mafb-deficient macrophages can be rescued by adding serum from wild-type mice, but not by adding serum from C1q-deficient mice. By hemolysis assay we also show that activation of the classical complement pathway is decreased in Mafb-deficient mice. In addition, MafB overexpression induces C1q-dependent gene expression and signals that induce C1q genes are less effective in the absence of MafB. We also show that Mafb-deficiency can increase glomerular autoimmunity, including anti-nuclear antibody deposition. These results show that MafB is an important regulator of C1q., Complement component C1q activates efferocytosis, suppresses inflammatory responses, and is thereby thought to limit autoimmune disease. Here, the authors show that macrophage transcription factor MafB regulates total serum levels of C1q, which contributes to preventing autoimmune disease in mice.

In order to increase the contribution of donor HSC cells, irradiation and DNA alkylating agents have been commonly used as experimental methods to eliminate HSCs for adult mice. But a technique of HSC deletion for mouse embryo for increase contribution of donor cells has not been published. Here, we established for the first time a procedure for placental HSC transplantation into E11.5 Runx1-deficient mice mated with G1-HRD-Runx1 transgenic mice (Runx1-/-::Tg mice) that have no HSCs in the fetal liver. Following the transplantation of fetal liver cells from mice (allogeneic) or rats (xenogeneic), high donor cell chimerism was observed in Runx1-/-::Tg embryos. Furthermore, chimerism analysis and colony assay data showed that donor fetal liver hematopoietic cells contributed to both white blood cells and red blood cells. Moreover, secondary transplantation into adult recipient mice indicated that the HSCs in rescued Runx1-/-::Tg embryos had normal abilities. These results suggest that mice lacking fetal liver HSCs are a powerful tool for hematopoiesis reconstruction during the embryonic stage and can potentially be used in basic research on HSCs or xenograft models.

Microglia are tissue-resident macrophages which are distributed throughout the central nervous system (CNS). Recent studies suggest that microglia are a unique myeloid population distinct from peripheral macrophages in terms of origin and gene expression signature. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a pleiotropic cytokine regulating myeloid development, has been shown to stimulate proliferation and alter phenotype of microglia in vitro. However, how its signaling is modulated in microglia is poorly characterized. MafB, a bZip transcriptional factor, is highly expressed in monocyte-macrophage lineage cells including microglia, although its role in microglia is largely unknown. We investigated the crosstalk between GM-CSF signaling and MafB by analyzing primary microglia. We found that Mafb-deficient microglia grew more rapidly than wild-type microglia in response to GM-CSF. Moreover, the expression of genes associated with microglial differentiation was more downregulated in Mafb-deficient microglia cultured with GM-CSF. Notably, such differences between the genotypes were not observed in the presence of M-CSF. In addition, we found that Mafb-deficient microglia cultured with GM-CSF barely extended their membrane protrusions, probably due to abnormal activation of RhoA, a key regulator of cytoskeletal remodeling. Altogether, our study reveals that MafB is a negative regulator of GM-CSF signaling in microglia. These findings could provide new insight into the modulation of cytokine signaling by transcription factors in microglia.

Macrophages manifest distinct phenotype according to the organs in which they reside. In addition, they flexibly switch their character in adaptation to the changing environment. However, the molecular basis that explains the conversion of the macrophage phenotype has so far been unexplored. We find that CD169+ macrophages change their phenotype by regulating the level of a transcription factor Maf both in vitro and in vivo in C57BL/6J mice. When CD169+ macrophages were exposed to bacterial components, they expressed an array of acute inflammatory response genes in Maf-dependent manner and simultaneously start to downregulate Maf. This Maf suppression is dependent on accelerated degradation through proteasome pathway and microRNA-mediated silencing. The downregulation of Maf unlocks the NF-E2–related factor 2–dominant, cytoprotective/antioxidative program in the same macrophages. The present study provides new insights into the previously unanswered question of how macrophages initiate proinflammatory responses while retaining their capacity to repair injured tissues during inflammation.

We report habenular lateralization in a simple transgenic mouse model used for labeling a facet of neuronal activity history. A transgenic construct comprised of a zif268/egr1 immediate-early gene promoter and a gene for normal Venus fluorescent protein with a membrane tag converted promoter activity into long-life fluorescent proteins, which was thought to describe a facet of neuronal activity history by summing neuronal activity. In addition to mapping the immediate-early gene-immunopositive cells, this method helped demonstrate the functionality of the lateral habenular nucleus (LHb). During postnatal development, the LHb was activated between postnatal days 10 and 16. The water-immersion restraint stress also activated the LHb over a similar period. LHb activation was functionally lateralized, but had no directional bias at the population level. Moreover, the posterior LHb was activated in the early stage after the stress, while the anterior LHb was activated in the later stage. Our results indicate lateralization, maturation, and anteroposterior topography of the LHb during postnatal development and the stress response.

Masculinization of external genitalia is an essential process in the formation of the male reproductive system. Prominent characteristics of this masculinization are the organ size and the sexual differentiation of the urethra. Although androgen is a pivotal inducer of the masculinization, the regulatory mechanism under the control of androgen is still unknown. Here, we address this longstanding question about how androgen induces masculinization of the embryonic external genitalia through the identification of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb) gene. Mafb is expressed prominently in the mesenchyme of male genital tubercle (GT), the anlage of external genitalia. MAFB expression is rarely detected in the mesenchyme of female GTs. However, exposure to exogenous androgen induces its mesenchymal expression in female GTs. Furthermore, MAFB expression is prominently down-regulated in male GTs of androgen receptor (Ar) KO mice, indicating that AR signaling is necessary for its expression. It is revealed that Mafb KO male GTs exhibit defective embryonic urethral formation, giving insight into the common human congenital anomaly hypospadias. However, the size of Mafb KO male GTs is similar with that of wild-type males. Moreover, androgen treatment fails to induce urethral masculinization of the GTs in Mafb KO mice. The current results provide evidence that Mafb is an androgen-inducible, sexually dimorphic regulator of embryonic urethral masculinization.

C1galt1 is essential for synthesis of the core 1 structure of mucin-type O-glycans. To clarify the physiological role of O-glycans in adult hematopoiesis, we exploited the interferon-inducible Mx1-Cre transgene to conditionally ablate the C1galt(flox) allele (Mx1-C1). Mx1-C1 mice exhibit severe thrombocytopenia, giant platelets, and prolonged bleeding times. Both the number and DNA ploidy of megakaryocytes in Mx1-C1 bone marrow were similar to those in wild-type (WT) mice. However, there were few proplatelets in Mx1-C1 primary megakaryocytes. Conversely, bone marrow transplanted from Mx1-C1 to WT and splenectomized Mx1-C1 mice gave rise to observations similar to those described above. The expression of GPIbα messenger RNA was unchanged in Mx1-C1 bone marrow, whereas flow cytometric and western blot analyses using megakaryocytes and platelets revealed that the expression of GPIbα protein was significantly reduced in Mx1-C1 mice. Moreover, circulating Mx1-C1 platelets exhibited an increase in the number of microtubule coils, despite normal levels of α- and β-tubulin. Our observations suggest that O-glycan is required for terminal megakaryocyte differentiation and platelet production and that the decrease in GPIbα in cells lacking O-glycan might be caused by increased proteolysis.

The choroid plexus (ChP) is a non-neural epithelial tissue that produces cerebrospinal fluid (CSF). The ChP differentiates from the roof plate, a dorsal midline structure of the neural tube. However, molecular mechanisms underlying ChP development are poorly understood compared to neural development. MafB is a bZip transcription factor that is known to be expressed in the roof plate. Here we investigated the role of MafB in embryonic development of the hindbrain ChP (hChP) using Mafb-deficient mice. Immunohistochemical analyses revealed that MafB is expressed in the roof plate and early hChP epithelial cells but its expression disappears at a later embryonic stage. We also found that the Mafb-deficient hChP exhibits delayed differentiation and results in hypoplasia compared to the wild-type hChP. Furthermore, the Mafb-deficient hChP exhibits increased apoptotic cell death and decreased proliferating cells at E12.5, an early stage of hChP development. Collectively, our findings reveal that MafB play an important role in promoting hChP development during embryogenesis.

Sepsis is a systemic inflammatory response to infection, accounting for the most common cause of death in intensive care units. Here, we report that peripheral administration of the hypothalamic neuropeptide orexin improves the survival of mice with lipopolysaccharide (LPS) induced endotoxin shock, a well-studied septic shock model. The effect is accompanied by a suppression of excessive cytokine production and an increase of catecholamines and corticosterone. We found that peripherally administered orexin penetrates the blood-brain barrier under endotoxin shock, and that central administration of orexin also suppresses the cytokine production and improves the survival, indicating orexin’s direct action in the central nervous system (CNS). Orexin helps restore body temperature and potentiates cardiovascular function in LPS-injected mice. Pleiotropic modulation of inflammatory response by orexin through the CNS may constitute a novel therapeutic approach for septic shock. DOI: http://dx.doi.org/10.7554/eLife.21055.001, eLife digest The body has a range of defenses to fight infection, which play a crucial role in keeping us healthy. However, sometimes the response to infection may damage the body’s own tissues and organs, leading to a life-threatening condition called sepsis. In the most severe stage of sepsis – known as septic shock – blood pressure drops to dangerously low levels and the individual often dies. There is currently no effective therapy for septic shock. Recent studies have revealed how the brain regulates immune responses via chemical signals and nerve impulses. A molecule called orexin is made in the brain and regulates the activity of a group of neurons that control sleep. Orexin can also alter heart rate and body temperature in rats, which suggests that it may have potential to be developed as a treatment for septic shock. To test this idea, Ogawa, Irukayama-Tomobe et al. injected orexin under the skin of mice with septic shock. The experiments show that the injected orexin is able to enter the brain, where it helps the mice to survive and recover from septic shock by restoring normal body temperature and boosting heart rate. Further experiments suggest that orexin is likely to regulate immune responses through multiple signaling pathways in the brain. The next step following on from this work is to find out the precise mechanism through which orexin regulates the responses of the immune system. This orexin treatment strategy should also be tested on primates with septic shock before planning any clinical trials in humans. DOI: http://dx.doi.org/10.7554/eLife.21055.002

MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains. Although it is well known that MafB is specifically expressed in glomerular epithelial cells (podocytes) and macrophages, characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions in the kidney and in macrophages, we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. MafB homozygous mutants displayed renal dysgenesis with abnormal podocyte differentiation as well as tubular apoptosis. Interestingly, these kidney phenotypes were associated with diminished expression of several kidney disease-related genes. In hematopoietic cells, GFP fluorescence was observed in both Mac-1- and F4/80-expressing macrophages in the fetal liver. Interestingly, F4/80 expression in macrophages was suppressed in the homozygous mutant, although development of the Mac-1-positive macrophage population was unaffected. In primary cultures of fetal liver hematopoietic cells, MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages, whereas the Mac-1-positive macrophage population developed normally. These results demonstrate that MafB is essential for podocyte differentiation, renal tubule survival, and F4/80 maturation in a distinct subpopulation of nonadherent mature macrophages.

The large Maf transcription factors c-Maf and MafB are expressed in macrophage-lineage hematopoietic cells, but the expression patterns of MafB and c-Maf in macrophage subtypes and tissue-resident macrophages have not been fully analyzed. First, we analyzed MafB and c-Maf protein expression in tissue-resident macrophages. Mouse lymph nodes, spleens, lungs, and kidneys were subjected to immunohistochemistry using anti-MafB and anti-c-Maf. Both MafB and c-Maf signals were observed in lymph node macrophages. In the splenic macrophages the MafB signal was detected by anti-MafB, but the c-Maf signal was not detected. No expression of c-Maf or MafB was detected in macrophages in the lung and kidney. Flow cytometry analysis revealed a similar pattern of GFP expression in Mafb/GFP knock-in heterozygous mice. To analyze these different expression patterns in greater detail, we examined the expression of MafB and c-Maf by quantitative RT-PCR in different cytokine- or LPS-induced macrophages in vitro. MafB expression was induced by IL-10 or IL-4 with IL-13 and was reduced by LPS or GM-CSF. By contrast, c-Maf expression was induced by IL-10 and reduced by IL-4 with IL-13 or GM-CSF. These results indicate that MafB and c-Maf have different expression patterns in macrophages, suggesting differences in function.

The mouse Flk1 gene is expressed in various mesodermal progenitor cells of developing embryos. Recent studies have shown that Flk1 expression marks multipotent mesodermal progenitors, giving rise to various hemato-cardiovascular cell lineages during development. Flk1 expression also marks hemato-cardiovascular cell lineages in differentiating embryonic stem (ES) cells, which may be used in transplantation decisions to treat cardiovascular diseases. Despite its developmental and clinical importance in cardiovascular tissues, the transcriptional regulatory system of Flk1 has remained unclear. Here, we report a novel enhancer of the mouse Flk1 gene directing early mesodermal expression during development as well as ES differentiation. The enhancer enriches various mesodermal progenitors, such as primitive erythropoietic progenitors, hemangioblast (BL-CFC) and cardiovascular progenitors (CV-CFC). The enhancer is activated by Bmp, Wnt and Fgf, and it contains Gata-, Cdx-, Tcf/Lef-, ER71/Etv2- and Fox-binding sites, some of which are bound specifically by each of these transcription factors. As these transcription factors are known to act under the control of the Bmp, Wnt and Fgf families, early Flk1 expression may be induced by cooperative interactions between Gata, Tcf/Lef, Cdx and ER71/Etv2 under the control of Bmp, Wnt and Fgf signaling. The enhancer is required for early Flk1 expression and for hemangioblast development during ES differentiation.

c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the activated protein-1 super family of basic leucine zipper proteins. Despite its overexpression in hematologic malignancies, the physiologic roles c-Maf plays in normal hematopoiesis have been largely unexplored. On a C57BL/6J background, c-Maf−/− embryos succumbed from severe erythropenia between embryonic day (E) 15 and E18. Flow cytometric analysis of fetal liver cells showed that the mature erythroid compartments were significantly reduced in c-Maf−/− embryos compared with c-Maf+/+ littermates. Interestingly, the CFU assay indicated there was no significant difference between c-Maf+/+ and c-Maf−/− fetal liver cells in erythroid colony counts. This result indicated that impaired definitive erythropoiesis in c-Maf−/− embryos is because of a non–cell-autonomous effect, suggesting a defective erythropoietic microenvironment in the fetal liver. As expected, the number of erythroblasts surrounding the macrophages in erythroblastic islands was significantly reduced in c-Maf−/− embryos. Moreover, decreased expression of VCAM-1 was observed in c-Maf−/− fetal liver macrophages. In conclusion, these results strongly suggest that c-Maf is crucial for definitive erythropoiesis in fetal liver, playing an important role in macrophages that constitute erythroblastic islands.

Multiple myeloma (MM) is a currently incurable neoplasm of terminally differentiated B cells. The translocation and/or overexpression of c-MAF have been observed in human MM. Although c-MAF might function as an oncogene in human MM, there has been no report thus far describing the direct induction of MM by c-MAF overexpression in vivo. In this study, we have generated transgenic (TG) mice that express c-Maf specifically in the B-cell compartment. Aged c-Maf TG mice developed B-cell lymphomas with some clinical features that resembled those of MM, namely, plasma cell expansion and hyperglobulinemia. Quantitative RT-PCR analysis demonstrated that Ccnd2 and Itgb7, which are known target genes of c-Maf, were highly expressed in the lymphoma cells. This novel TG mouse model of the human MM t(14;16)(q32;q23) chromosomal translocation should serve to provide new insight into the role of c-MAF in tumorigenesis. Cancer Res; 71(2); 339–48. ©2011 AACR.

During vertebrate lens development, the anterior, ectoderm-derived lens vesicle cells differentiate into a monolayer of epithelial cells that retain proliferative potential. Subsequently, they exit the cell cycle and give rise to posterior lens fiber cells that form the lens body. In the present study, we demonstrate that the transcription factor GATA-3 is expressed in the posterior lens fiber cells during embryogenesis, and that GATA-3 deficiency impairs lens development. Interestingly, expression of E-cadherin, a premature lens vesicle marker, is abnormally prolonged in the posterior region of Gata3 homozygous mutant lenses. Furthermore, expression of gamma-crystallin, a differentiation marker for fiber cells, is reduced. This suppressed differentiation is accompanied by an abnormal cellular proliferation, as well as with diminished levels of the cell-cycle inhibitors Cdkn1b/p27 and Cdkn1c/p57 and increased Ccnd2/cyclin D2 abundance. Thus, these observations suggest that GATA-3 is essential for lens cells differentiation and proper cell cycle control.

Mammalian epidermis is a stratified epithelium composed of distinct layers of keratinocytes. The outermost cornified layer is a primary barrier that consists of a cornified envelope, an insoluble structure assembled by cross-linked scaffold proteins, and a surrounding mixture of lipids. Skin keratinocytes undergo a multistep differentiation process, but the mechanism underlying this process is not fully understood. We demonstrate that the transcription factor MafB is expressed in differentiating keratinocytes in mice and is transcriptionally upregulated upon human keratinocyte differentiation in vitro. In MafB-deficient mice, epidermal differentiation was partially impaired and the cornified layer was thinner than in wild-type mice. On the basis of transcriptional profiling, we detected reduced expression levels of a subset of cornified envelope genes, for example, filaggrin and repetin, in the MafB(-/-) epidermis. By contrast, the expression levels of lipid metabolism-related genes, such as Alox12e and Smpd3, increased. The upregulated genes in the MafB(-/-) epidermis were enriched for putative target genes of the transcription factors Gata3, Grhl3, and Klf4. Immunohistochemical analysis of skin biopsy samples revealed that the expression levels of filaggrin and MafB were significantly reduced in patients with human atopic dermatitis and psoriasis vulgaris. Our results indicate that MafB is a component of the gene expression program that regulates epidermal keratinocyte differentiation.

MafA is a transcription factor that binds to the promoter in the insulin gene and has been postulated to regulate insulin transcription in response to serum glucose levels, but there is no current in vivo evidence to support this hypothesis. To analyze the role of MafA in insulin transcription and glucose homeostasis in vivo, we generated MafA-deficient mice. Here we report that MafA mutant mice display intolerance to glucose and develop diabetes mellitus. Detailed analyses revealed that glucose-, arginine-, or KCl-stimulated insulin secretion from pancreatic beta cells is severely impaired, although insulin content per se is not significantly affected. MafA-deficient mice also display age-dependent pancreatic islet abnormalities. Further analysis revealed that insulin 1, insulin 2, Pdx1, Beta2, and Glut-2 transcripts are diminished in MafA-deficient mice. These results show that MafA is a key regulator of glucose-stimulated insulin secretion in vivo.

MafB is a transcription factor that induces myelomonocytic differentiation. However, the precise role of MafB in the pathogenic function of macrophages has never been clarified. Here we demonstrate that MafB promotes hyperlipidemic atherosclerosis by suppressing foam-cell apoptosis. Our data show that MafB is predominantly expressed in foam cells found within atherosclerotic lesions, where MafB mediates the oxidized LDL-activated LXR/RXR-induced expression of apoptosis inhibitor of macrophages (AIM). In the absence of MafB, activated LXR/RXR fails to induce the expression of AIM, a protein that is normally responsible for protecting macrophages from apoptosis; thus, Mafb-deficient macrophages are prone to apoptosis. Haematopoietic reconstitution with Mafb-deficient fetal liver cells in recipient LDL receptor-deficient hyperlipidemic mice revealed accelerated foam-cell apoptosis, which subsequently led to the attenuation of the early atherogenic lesion. These findings represent the first evidence that the macrophage-affiliated MafB transcription factor participates in the acceleration of atherogenesis.

形態: カラー図版あり, Physical characteristics: Original contains color illustrations, Accepted: 2017-08-17, 資料番号: PA1810019000

Retinoic acid related orphan receptor gamma-t (RORγt) is known to be a master regulator of Th17-cell development. In this study, we generated RORγt-overexpressing transgenic (RORγt Tg) mice in which transgene expression was driven by the CD2 promoter, and found that these mice developed polyclonal plasmacytosis and autoantibody production. RORγt Tg mice were generated on a C57BL/6 background, and also were intercrossed with BALB/c mice. BALB/c F1 (BALB/F1) RORγt Tg mice developed massive polyclonal plasma-cytosis, and had shorter life spans. Splenomegaly and infiltration of plasma cells into the lung were observed. Hyperglobulinemia, anti-double-stranded DNA antibodies, anti-erythrocyte antibodies, and anti-platelet antibodies were detected in BALB/F1 RORγt Tg mice. In the present study, polyclonal plasmacytosis in BALB/F1 RORγt Tg mice appeared to be due to the induction of excessive IL-6 production by IL-17. We detected increased numbers of CD11b(+) cells that produced IL-6. We also generatedIL-6-deficient RORγt Tg BALB/F1 background mice, which displayed high levels of serum IL-17, but did not develop severe hyperglobulinemia. Excessive IL-6 production by several cell types, including macrophages, in BALB/F1 RORγt Tg mice, might effect the development of plasma-cytosis. These results suggest that RORγt plays important roles in the development of plasmacytosis and autoantibody production.

Serum calcium and phosphate homeostasis is critically regulated by parathyroid hormone (PTH) secreted by the parathyroid glands. Parathyroid glands develop from the bilateral parathyroid-thymus common primordia. In mice, the expression of transcription factor Glial cell missing 2 (Gcm2) begins in the dorsal/anterior part of the primordium on embryonic day 9.5 (E9.5), specifying the parathyroid domain. The parathyroid primordium then separates from the thymus primordium and migrates to its adult location beside the thyroid gland by E15.5. Genetic ablation of gcm2 results in parathyroid agenesis in mice, indicating that Gcm2 is essential for early parathyroid organogenesis. However, the regulation of parathyroid development at later stages is not well understood. Here we show that transcriptional activator v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (MafB) is developmentally expressed in parathyroid cells after E11.5. MafB expression was lost in the parathyroid primordium of gcm2 null mice. The parathyroid glands of mafB(+/-) mice were mislocalized between the thymus and thyroid. In mafB(-/-) mice, the parathyroid did not separate from the thymus. Furthermore, in mafB(-/-) mice, PTH expression and secretion were impaired; expression levels of renal cyp27b1, one of the target genes of PTH, was decreased; and bone mineralization was reduced. We also demonstrate that although Gcm2 alone does not stimulate the PTH gene promoter, it associates with MafB to synergistically activate PTH expression. Taken together, our results suggest that MafB regulates later steps of parathyroid development, that is, separation from the thymus and migration toward the thyroid. MafB also regulates the expression of PTH in cooperation with Gcm2.

Aging leads to the disruption of the homeostatic balance of multiple biological systems. In bone marrow multipotent mesenchymal cells undergo differentiation into various anchorage-dependent cell types, including osteoblasts and adipocytes. With age as well as with treatment of antidiabetic drugs such as thiazolidinediones, mesenchymal cells favor differentiation into adipocytes, resulting in an increased number of adipocytes and a decreased number of osteoblasts, causing osteoporosis. The mechanism behind this differentiation switch is unknown. Here we show an age-related decrease in the expression of Maf in mouse mesenchymal cells, which regulated mesenchymal cell bifurcation into osteoblasts and adipocytes by cooperating with the osteogenic transcription factor Runx2 and inhibiting the expression of the adipogenic transcription factor Pparg. The crucial role of Maf in both osteogenesis and adipogenesis was underscored by in vivo observations of delayed bone formation in perinatal Maf(-/-) mice and an accelerated formation of fatty marrow associated with bone loss in aged Maf(+/-) mice. This study identifies a transcriptional mechanism for an age-related switch in cell fate determination and may provide a molecular basis for novel therapeutic strategies against age-related bone diseases.

Background The hair follicle of mammalian skin consists of a group of concentric epithelial cell layers. The inner root sheath (IRS), which surrounds the hardening hair shaft beneath the skin surface, is subdivided into three layers, termed the cuticle of the IRS, Huxley's layer, and Henle's layer. The IRS forms a follicular wall in the hair canal and helps guide the developing hair shaft. c-Maf and MafB, members of the Maf family of transcription factors, play important roles in the developmental processes of various tissues and in cell type-specific gene expression. Objective The aim of this study is to reveal the pattern of expression and functional roles of c-Maf and MafB in the hair follicle. Methods We determined the precise location of c-Maf and MafB expression using immunofluorescent staining of mouse skin sections with layer-specific markers. We also analyzed whiskers of c- maf - and mafB -null mice (c- maf −/− and mafB −/− , respectively) using scanning electron microscopy. Results c-Maf and MafB were differentially expressed in the Huxley's and Henle's layers of the IRS. Scanning electron microscopic analysis showed irregular cuticle patterning of whiskers of c- maf −/− and mafB −/− mice. The cuticles of mafB −/− mice were also thinner than those of wild-type mice. Conclusion c-Maf and MafB are expressed in the IRS layers in a lineage-restricted manner and are involved in hair morphogenesis.

The importance of the large Maf transcription factor family has been investigated in lens development in the chick, Xenopus and mammals. Previously we reported that c-maf-deficient mice exhibit severe defects in lens fibre cells. Here, we report the roles of other large Mafs, MafA/L-Maf and MafB, during mouse lens development. MafA/L-Maf and MafB were expressed in lens epithelial cells and fibre cells at E12.5 but had largely disappeared from the lens at E18.5. The lens of mafA-, mafB-deficient and mafA::mafB double-deficient mice developed normally. In c-maf-deficient mice, the pattern of expression of MafA and MafB differed from their expression in wild-type mice. Moreover, the expression of crystallin genes was unchanged in mafA-, mafB- and mafA::mafB double-deficient lens. These results indicate that c-Maf alone is essential for lens development, and that MafA/L-Maf and MafB are dispensable in mice.

application/pdf, c-Maf, which is one of the large Maf transcription factors, can bind to Maf recognition element (MARE) and activates transcription of target genes. Although c-Maf is expressed in macrophages and directly regulates the expression of interleukin-10, detailed information regarding its function in the null mutant phenotype of tissue macrophages remain unknown. In this study, we demonstrated that c-Maf is specifically expressed in the F4/80 positive fetal liver and adult macrophages. The expression of F4/80, which is a tissue macrophage-specific seven trans-membrane receptor, was dramatically suppressed in the c-Maf-deficient macrophage, whereas the expression of Mac-1 was not affected, suggesting that c-Maf is not necessary for the lineage commitment of macrophages. Luciferase reporter and EMSA showed that c-Maf directly regulates the expression of F4/80 by interacting with the half-MARE site of the F4/80 promoter. These results suggest that c-Maf is required for the F4/80 expression in macrophages in vivo.

Gata3 mutant mice expire of noradrenergic deficiency by embryonic day (E) 11 and can be rescued pharmacologically or, as shown here, by restoring Gata3 function specifically in sympathoadrenal (SA) lineages using the human DBH promoter to direct Gata3 transgenic expression. In Gata3-null embryos, there was significant impairment of SA differentiation and increased apoptosis in adrenal chromaffin cells and sympathetic neurons. Additionally, mRNA analyses of purified chromaffin cells from Gata3 mutants show that levels of Mash1, Hand2 and Phox2b(postulated upstream regulators of Gata3) as well as terminally differentiated SA lineage products (tyrosine hydroxylase, Th, and dopamineβ-hydroxylase, Dbh) are markedly altered. However, SA lineage-specific restoration of Gata3 function in the Gata3 mutant background rescues the expression phenotypes of the downstream, as well as the putative upstream genes. These data not only underscore the hypothesis that Gata3 is essential for the differentiation and survival of SA cells, but also suggest that their differentiation is controlled by mutually reinforcing feedback transcriptional interactions between Gata3, Mash1, Hand2 and Phox2b in the SA lineage.

The b-Zip transcription factor MafB is an essential determinant of neural development and an inducer of monocytic differentiation. The MafB protein is expressed in a variety of tissues including the developing spinal cord, retina, myelomonocytic lineages of hematopoietic cells, and peritoneal macrophages. However, the tissue-specific regulatory mechanism of mafB gene expression and its biological relevance have not been examined in detail. Here, we report, for the first time, analysis of the regulatory mechanism and tissue-specific expression of the mafB gene in vivo using transgenic mice. A transgene, containing the 8.2-kb sequence flanking the 5' end of the mafB exon, directed the expression of a GFP reporter gene specifically in the retina, myelomonocytic lineages of hematopoietic cells, peritoneal macrophages and the ventral spinal cord. In situ hybridization analysis showed that the reporter gene expression specifically recapitulates the endogenous expression profile of mafB in the retina and spinal cord. FACS analysis revealed that the Gr-1, Mac-1 and F4/80 antigens were present on most of the GFP-positive hematopoietic cells from transgenic adult bone marrow and spleen. On the other hand, B220 CD4, 8, and Ter119 cells were almost absent from among the GFP-positive cells examined. These observations suggest that gene regulatory regions located in the 8.2-kb upstream region of mafB are responsible for directing mafB expression in the retina, myelomonocytic lineages, peritoneal macrophages and the ventral spinal cord.

Background Deficiency of the transcription factor MafB, which is normally expressed in macrophages, can underlie cellular dysfunction associated with a range of autoimmune diseases and arteriosclerosis. MafB has important roles in cell differentiation and regulation of target gene expression; however, the mechanisms of this regulation and the identities of other transcription factors with which MafB interacts remain uncertain. Bioinformatics methods provide a valuable approach for elucidating the nature of these interactions with transcriptional regulatory elements from a large number of DNA sequences. In particular, identification of patterns of co-occurrence of regulatory cis-elements (motifs) offers a robust approach. Results Here, the directional relationships among several functional motifs were evaluated using the Log-linear Graphical Model (LGM) after extraction and search for evolutionarily conserved motifs. This analysis highlighted GATA-1 motifs and 5’AT-rich half Maf recognition elements (MAREs) in promoter regions of 18 genes that were down-regulated in Mafb deficient macrophages. GATA-1 motifs and MafB motifs could regulate expression of these genes in both a negative and positive manner, respectively. The validity of this conclusion was tested with data from a luciferase assay that used a C1qa promoter construct carrying both the GATA-1 motifs and MAREs. GATA-1 was found to inhibit the activity of the C1qa promoter with the GATA-1 motifs and MafB motifs. Conclusions These observations suggest that both the GATA-1 motifs and MafB motifs are important for lineage specific expression of C1qa. In addition, these findings show that analysis of combinations of evolutionarily conserved motifs can be successfully used to identify patterns of gene regulation.