Hitoshi Miyachi

MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity

Adipocytes secrete a variety of bioactive molecules that affect the insulin sensitivity of other tissues. We now show that the abundance of monocyte chemoattractant protein-1 (MCP-1) mRNA in adipose tissue and the plasma concentration of MCP-1 were increased both in genetically obese diabetic (db/db) mice and in WT mice with obesity induced by a high-fat diet. Mice engineered to express an MCP-1 transgene in adipose tissue under the control of the aP2 gene promoter exhibited insulin resistance, macrophage infiltration into adipose tissue, and increased hepatic triglyceride content. Furthermore, insulin resistance, hepatic steatosis, and macrophage accumulation in adipose tissue induced by a high-fat diet were reduced extensively in MCP-1 homozygous KO mice compared with WT animals. Finally, acute expression of a dominant-negative mutant of MCP-1 ameliorated insulin resistance in db/db mice and in WT mice fed a high-fat diet. These findings suggest that an increase in MCP-1 expression in adipose tissue contributes to the macrophage infiltration into this tissue, insulin resistance, and hepatic steatosis associated with obesity in mice.

Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET.

Endogenous retroviruses (ERVs), retrovirus-like elements with long terminal repeats, are widely dispersed in the euchromatic compartment in mammalian cells, comprising ∼10% of the mouse genome. These parasitic elements are responsible for >10% of spontaneous mutations. Whereas DNA methylation has an important role in proviral silencing in somatic and germ-lineage cells, an additional DNA-methylation-independent pathway also functions in embryonal carcinoma and embryonic stem (ES) cells to inhibit transcription of the exogenous gammaretrovirus murine leukaemia virus (MLV). Notably, a recent genome-wide study revealed that ERVs are also marked by histone H3 lysine 9 trimethylation (H3K9me3) and H4K20me3 in ES cells but not in mouse embryonic fibroblasts. However, the role that these marks have in proviral silencing remains unexplored. Here we show that the H3K9 methyltransferase ESET (also called SETDB1 or KMT1E) and the Krüppel-associated box (KRAB)-associated protein 1 (KAP1, also called TRIM28) are required for H3K9me3 and silencing of endogenous and introduced retroviruses specifically in mouse ES cells. Furthermore, whereas ESET enzymatic activity is crucial for HP1 binding and efficient proviral silencing, the H4K20 methyltransferases Suv420h1 and Suv420h2 are dispensable for silencing. Notably, in DNA methyltransferase triple knockout (Dnmt1-/-Dnmt3a-/-Dnmt3b-/-) mouse ES cells, ESET and KAP1 binding and ESET-mediated H3K9me3 are maintained and ERVs are minimally derepressed. We propose that a DNA-methylation-independent pathway involving KAP1 and ESET/ESET-mediated H3K9me3 is required for proviral silencing during the period early in embryogenesis when DNA methylation is dynamically reprogrammed.

Oscillatory Control of Factors Determining Multipotency and Fate in Mouse Neural Progenitors

Oscillation Stabilizes the Progenitor State Transcription factors regulate fate choice between different neural lineages, but the same transcription factors are also expressed in neural progenitor cells. Imayoshi et al. (p. 1203, published online 31 October) analyzed the details of expression of several transcription factors in mouse neural cells. In neural progenitor cells, several different transcription factors were expressed in an oscillatory manner, whereas differentiated neurons stably expressed a single lineage-specific factor. During neural development, the differentiated state correlates with sustained expression of a single fate-determination factor. The basic helix-loop-helix transcription factors Ascl1/Mash1, Hes1, and Olig2 regulate fate choice of neurons, astrocytes, and oligodendrocytes, respectively. These same factors are coexpressed by neural progenitor cells. Here, we found by time-lapse imaging that these factors are expressed in an oscillatory manner by mouse neural progenitor cells. In each differentiation lineage, one of the factors becomes dominant. We used optogenetics to control expression of Ascl1 and found that, although sustained Ascl1 expression promotes neuronal fate determination, oscillatory Ascl1 expression maintains proliferating neural progenitor cells. Thus, the multipotent state correlates with oscillatory expression of several fate-determination factors, whereas the differentiated state correlates with sustained expression of a single factor.

Epigenetic Regulation of Mouse Sex Determination by the Histone Demethylase Jmjd1a

More Determined Sex Although several transcription factors participate in mammalian sex determination, the contribution from specific epigenetic regulation is just being revealed. Kuroki et al. (p. 1106) show that a JmjC domain–containing protein, Jmjd1a, catalyzes H3K9 demethylation of the Y-linked sex-determining gene Sry in mice to enable its expression above the required threshold level. Ablation of Jmjd1a function results in mouse male-to-female sex reversal, hence not only revealing a mechanism of Sry regulation but also the pivotal role of epigenetic regulation in mammalian sex determination. Histone modification controls mammalian sex determination. Developmental gene expression is defined through cross-talk between the function of transcription factors and epigenetic status, including histone modification. Although several transcription factors play crucial roles in mammalian sex determination, how epigenetic regulation contributes to this process remains unknown. We observed male-to-female sex reversal in mice lacking the H3K9 demethylase Jmjd1a and found that Jmjd1a regulates expression of the mammalian Y chromosome sex-determining gene Sry. Jmjd1a directly and positively controls Sry expression by regulating H3K9me2 marks. These studies reveal a pivotal role of histone demethylation in mammalian sex determination.

Intronic delay is essential for oscillatory expression in the segmentation clock

Proper timing of gene expression is essential for many biological events, but the molecular mechanisms that control timing remain largely unclear. It has been suggested that introns contribute to the timing mechanisms of gene expression, but this hypothesis has not been tested with natural genes. One of the best systems for examining the significance of introns is the oscillator network in the somite segmentation clock, because mathematical modeling predicted that oscillating expression depends on negative feedback with a delayed timing. The basic helix–loop–helix repressor gene Hes7 is cyclically expressed in the presomitic mesoderm (PSM) and regulates the somite segmentation. Here, we found that introns lead to an ∼19-min delay in the Hes7 gene expression, and mathematical modeling suggested that without such a delay, Hes7 oscillations would be abolished. To test this prediction, we generated mice carrying the Hes7 locus whose introns were removed. In these mice, Hes7 expression did not oscillate but occurred steadily, leading to severe segmentation defects. These results indicate that introns are indeed required for Hes7 oscillations and point to the significance of intronic delays in dynamic gene expression.

Identification of IL-7–Producing Cells in Primary and Secondary Lymphoid Organs Using IL-7–GFP Knock-In Mice

IL-7 is a cytokine crucial for development and maintenance of lymphocytes and other hematopoietic cells. However, how IL-7–expressing cells are distributed in lymphoid organs is not well known. To address this question, we established and analyzed IL-7–GFP knock-in mice. Thymic epithelial cells (TECs) expressed high GFP levels in the cortex and medulla, as detected with an anti-GFP Ab. Thymic mesenchymal cells also expressed GFP. Flow cytometry analysis suggested that cortical TECs expressed higher GFP levels than did medullary TECs. In bone marrow, immunohistochemistry indicated high levels of GFP in many VCAM-1+ mesenchymal stromal cells and in some VCAM-1− cells. Additionally, half of the VCAM-1+CD31− stromal cells and some platelet-derived growth factor receptor α+ stromal cells were GFP+, as detected by flow cytometry. Moreover, we detected GFP expression in fibroblastic reticular cells in the T cell zone and cortical ridge of lymph nodes. Remarkably, lymphatic endothelial cells (LECs) expressed GFP at high levels within the lymph node medulla, skin epidermis, and intestinal tissues. Additionally, we detected abundant IL-7 transcripts in isolated LECs, suggesting that LECs produce IL-7, a heretofore unknown finding. Furthermore, GFP is expressed in a subpopulation of intestinal epithelial cells, and that expression was markedly upregulated in a dextran sulfate sodium-induced acute colitis model. Overall, IL-7–GFP knock-in mice serve as a unique and powerful tool to examine the identity and distribution of IL-7–expressing cells in vivo.

Characterization of the IL-15 niche in primary and secondary lymphoid organs in vivo

Significance IL-15 is a cytokine critical for development and maintenance of T lymphoid cells. However, the identity and distribution of IL-15–expressing cells in lymphoid organs are not well understood. The present study reveals, by using IL-15–CFP knock-in mice that IL-15 was expressed in subsets of thymic epithelial cells, bone marrow stromal cells, lymph node stromal cells, and blood endothelial cells, a unique perspective of IL-15 niche in immune microenvironment. Taken together with our previous observation on IL-7–producing cells, this study suggests that some stromal cells express IL-7 and IL-15 differentially. Thus, the immune microenvironment appears to be consisted of functionally distinct subsets of stromal cells, expressing different cytokines. IL-15 is a cytokine critical for development, maintenance, and response of T cells, natural killer (NK) cells, NK T cells, and dendritic cells. However, the identity and distribution of IL-15–expressing cells in lymphoid organs are not well understood. To address these questions, we established and analyzed IL-15–CFP knock-in mice. We found that IL-15 was highly expressed in thymic medulla, and medullary thymic epithelial cells with high MHC class II expression were the major source of IL-15. In bone marrow, IL-15 was detected primarily in VCAM-1+PDGFRβ+CD31−Sca-1− stromal cells, which corresponded to previously described CXCL12-abundant reticular cells. In lymph nodes, IL-15–expressing cells were mainly distributed in the T-cell zone and medulla. IL-15 was expressed in some fibroblastic reticular cells and gp38−CD31− double-negative stromal cells in the T-cell zone. Blood endothelial cells, including all high endothelial venules, also expressed high IL-15 levels in lymph nodes, whereas lymphatic endothelial cells (LECs) lacked IL-15 expression. In spleen, IL-15 was expressed in VCAM-1+ stromal cells, where its expression increased as mice aged. Finally, IL-15 expression in blood and LECs of peripheral lymphoid organs significantly increased in LPS-induced inflammation. Overall, we have identified and characterized several IL-15–expressing cells in primary and secondary lymphoid organs, providing a unique perspective of IL-15 niche in immune microenvironment. This study also suggests that some stromal cells express IL-7 and IL-15 differentially and suggests a way to functionally classify different stromal cell subsets.

Zinc-finger gene Fez in the olfactory sensory neurons regulates development of the olfactory bulb non-cell-autonomously

Fez is a zinc-finger gene encoding a transcriptional repressor that is expressed in the olfactory epithelium, hypothalamus, ventrolateral pallium and prethalamus at mid-gestation. To reveal its function, we generated Fez-deficient mice. The Fez-deficient mice showed several abnormalities in the olfactory system: (1) impaired axonal projection of the olfactory sensory neurons; (2) reduced size of the olfactory bulb; (3) abnormal layer formation in the olfactory bulb; and (4) aberrant rostral migration of the interneuron progenitors. Fez was not expressed in the projection neurons, interneurons or interneuron progenitors. Transgene-mediated expression of Fez in olfactory sensory neurons significantly rescued the abnormalities in olfactory axon projection and in the morphogenesis of the olfactory bulb in Fez-knockout mice. Thus, Fez is cell-autonomously required for the axon termination of olfactory sensory neurons, and Fez non-cell-autonomously controls layer formation and interneuron development in the olfactory bulb. These findings suggest that signals from olfactory sensory neurons contribute to the proper formation of the olfactory bulb.

Continuous Postnatal Neurogenesis Contributes to Formation of the Olfactory Bulb Neural Circuits and Flexible Olfactory Associative Learning

The olfactory bulb (OB) is one of the two major loci in the mammalian brain where newborn neurons are constantly integrated into the neural circuit during postnatal life. Newborn neurons are generated from neural stem cells in the subventricular zone (SVZ) of the lateral ventricle and migrate to the OB through the rostral migratory stream. The majority of these newborn neurons differentiate into inhibitory interneurons, such as granule cells and periglomerular cells. It has been reported that prolonged supply of newborn neurons leads to continuous addition/turnover of the interneuronal populations and contributes to functional integrity of the OB circuit. However, it is not still clear how and to what extent postnatal-born neurons contribute to OB neural circuit formation, and the functional role of postnatal neurogenesis in odor-related behaviors remains elusive. To address this question, here by using genetic strategies, we first determined the unique integration mode of newly born interneurons during postnatal development of the mouse OB. We then manipulated these interneuron populations and found that continuous postnatal neurogenesis in the SVZ-OB plays pivotal roles in flexible olfactory associative learning and memory.

TRIC-A Channels in Vascular Smooth Muscle Contribute to Blood Pressure Maintenance.

TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation channels postulated to mediate counter-ion movements facilitating physiological Ca(2+) release from internal stores. Tric-a-knockout mice developed hypertension during the daytime due to enhanced myogenic tone in resistance arteries. There are two Ca(2+) release mechanisms in vascular smooth muscle cells (VSMCs); incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization, while agonist-induced activation of inositol trisphosphate receptors (IP(3)Rs) evokes global Ca(2+) transients causing contraction. Tric-a gene ablation inhibited RyR-mediated hyperpolarization signaling to stimulate voltage-dependent Ca(2+) influx, and adversely enhanced IP(3)R-mediated Ca(2+) transients by overloading Ca(2+) stores in VSMCs. Moreover, association analysis identified single-nucleotide polymorphisms (SNPs) around the human TRIC-A gene that increase hypertension risk and restrict the efficiency of antihypertensive drugs. Therefore, TRIC-A channels contribute to maintaining blood pressure, while TRIC-A SNPs could provide biomarkers for constitutional diagnosis and personalized medical treatment of essential hypertension.