Shinobu Tamura

Foxp1 gene expression in projection neurons of the mouse striatum

The developmental processes of maturation in the CNS are the result of specific events including mitogenesis, differentiation, and cell death which occur in a precise spatial and temporal manner. It has been reported that many transcription factors, including forkhead transcription factors, play a key role in these processes. First, we examined the expression pattern of the forkhead transcription factor Foxp1 in the adult CNS. Foxp1 was highly expressed in the striatum and moderately in the cerebral cortex, CA1/2 subfields of the hippocampus, and several thalamic nuclei. In situ hybridization combined with immunohistochemistry in the striatum of adult mice revealed that Foxp1 mRNA was detected in a subset of projection neurons, not in interneurons. In addition, the expression of Foxp1 mRNA was observed in the developing basal ganglia with the exception of the globus pallidus. Thus, Foxp1 mRNA was expressed in a subset of striatal projection neurons, probably the matrix neurons. The expression pattern of Foxp1 mRNA suggests that Foxp1 may play a role in the development and formation of a circuit in the basal ganglia, which is involving the matrix neurons.

Essential function of oncostatin m in nociceptive neurons of dorsal root ganglia.

Oncostatin M (OSM) is a member of the interleukin-6 family of cytokines, and we have reported previously that the murine OSM receptor β subunit (OSMR) was expressed in some neurons in the adult trigeminal and dorsal root ganglia (DRGs) and in the perineonatal hypoglossal nucleus. In the present study, we investigated the development of OSMR-positive neurons of DRGs in OSM-deficient mice. In situ hybridization revealed that OSMR-positive neurons in DRGs began to appear at postnatal day 0 (P0) and reached the adult level at P14. In the DRGs of the OSM-deficient mice, vanilloid receptor 1 (VR1)- and P2X3-positive small-sized neurons were significantly decreased. In addition, OSMR-positive neurons decreased, resulting in the reduced number of VR1/P2X3/OSMR-triple positive neurons. OSM-deficient mice displayed significantly reduced noxious responses in models of acute thermal, mechanical, chemical, and visceral pain. Thus, OSM plays an essential role in the development of a subtype of nociceptive neurons in the DRGs.

Expression of oncostatin M receptor β in a specific subset of nociceptive sensory neurons

Oncostatin M belongs to the interleukin‐6 family of cytokines and acts as a multifunctional cytokine during murine embryogenesis and in inflammatory reactions. Although it has been demonstrated that oncostatin M has biological activities on many types of cells, including hepatocytes, dermal fibroblasts and endothelial cells, the roles of oncostatin M in the murine peripheral nervous system remain unclear. Here, we investigated the expression of specific β‐subunit of oncostatin M receptor in the dorsal root ganglia of adult mice. In the adult dorsal root ganglia, β‐subunit of oncostatin M receptor was exclusively expressed in small‐sized neurons. Approximately 13% of total dorsal root ganglia neurons in mice contained β‐subunit of oncostatin M receptor. The double‐immunofluorescence method revealed that approximately 28% of β‐subunit of oncostatin M receptor‐positive neurons contained TrkA immunoreactivity, 63% expressed Ret immunoreactivity and 58% bound isolectin B4. No neuropeptides, including substance P and calcitonin gene‐related peptide, were contained in the neurons. In addition, all β‐subunit of oncostatin M receptor‐positive neurons expressed both vanilloid receptor 1 and P2X3 purinergic receptor. These neurons projected to the inner portion of lamina II in the dorsal horn of spinal cord and the dermis of skin. Seven days after sciatic nerve axotomy, the expression of β‐subunit of oncostatin M receptor was down‐regulated in the lumbar dorsal root ganglia of the injured side. Our study demonstrated that β‐subunit of oncostatin M receptor was expressed in both cell bodies and processes of nonpeptidergic nociceptive neurons in adult mice, suggesting that oncostatin M may affect the nociceptive function of the neurons through the modulation of vanilloid receptor 1 and P2X3 expression.

TRPV2, a capsaicin receptor homologue, is expressed predominantly in the neurotrophin-3-dependent subpopulation of primary sensory neurons

TRPV2, a member of transient receptor potential ion channels, responds to high-threshold noxious heat, but neither to capsaicin nor to proton. Although TRPV2 is expressed in medium- to large-sized dorsal root ganglion (DRG) neurons with myelinated fibers in adult rodents, little is known about the neurotrophin dependence of TRPV2-positive neurons in the developing and adult DRGs of mice. In the present study, using immunohistochemistry, we found that TRPV2 was first expressed in DRG neurons at embryonic day (E) 11.5, when neither TRPV1 nor TRPM8 was detected yet. Double-immunofluorescence staining revealed that tyrosine kinase receptor C (TrkC) was expressed in most of TRPV2-positive DRG neurons at E11.5 and E13.5. In addition, the percentage of TRPV2-positive neurons in the total DRG neurons at E13.5 reached the same as that of adulthood. In adult DRGs, TrkC and Ret were expressed in 68% and 25% of TRPV2-positive neurons, respectively. These results suggest that TRPV2 is expressed predominantly in the NT-3-dependent subpopulation of DRG neurons throughout development and in adult mice.

Expression pattern of the winged-helix/forkhead transcription factor Foxp1 in the developing central nervous system

The winged-helix/forkhead transcription factor gene family has been shown to play important roles in the development of the central nervous system (CNS) as well as heart, lung, and liver. Recently, we have identified Foxp1, a novel subfamily of winged-helix/forkhead genes, which was abundant in the lung and brain of adult mice. Here we analyzed the expression pattern of Foxp1 in the developing CNS using in situ hybridization. The expression of Foxp1 mRNA was first detected in the ventral horn of the spinal cord at 9.5 days postcoitum. During the late-stage of development, its gene expression was not detectable in neuroepithelia, but was clearly observed in the postmitotic neurons of various CNS regions, including caudate-putamen, neocortex, several brainstem nuclei, and cerebellum. In neonates, its gene expression was persisted in these motor-related regions.

Developmental expression pattern of oncostatin M receptor β in mice

Oncostatin M (OSM), which is predominantly expressed in bone marrow, is a member of the interleukin-6 family of cytokines, and appears to play important roles in hematopoiesis and the development of the liver. Recently, specific beta subunit of OSM receptor (OSMRbeta) was isolated from LO cells originated from aorta-gonad-mesonephros (AGM) region. In this study, we performed in situ hybridization to explore the expression pattern of OSMRbeta during murine embryogenesis, postnatal development, and in adult tissues. At 11.5 days postcoitum (dpc), the expression of OSMRbeta was first detected in aortic endothelial cells of the AGM region. At 14.5dpc, its gene expression was clearly observed in the primordia of some organs, including liver, thymus, choroid plexus, and limb, and persisted into postnatal mice. After birth, its gene expression became detectable in the other organs, such as lymph node, bone, heart, kidney, small intestine, nasal cavity, and lung.

Expression of oncostatin M in hematopoietic organs

Murine oncostatin M (OSM), a member of the interleukin‐6 (IL‐6) ‐related cytokine subfamily, stimulates definitive hematopoiesis and liver development. The OSM gene was cloned as a cytokine‐inducible early response gene in some hematopoietic cell lines. In this study, we performed in situ hybridization to examine the tissue distribution of cells expressing OSM mRNA in the developing and the adult mice. Its gene expression was seen in hematopoietic cells of developing liver from 11.5 days postcoitum (dpc), and persisted to the neonates. From 17.5 dpc, OSM mRNA‐positive cells were found in other hematopoietic organs, including bone marrow, thymus, and spleen. The highest levels of gene expression were observed in the adult bone marrow. Most OSM‐expressing cells expressed IL‐5 receptor α subunit, a marker for eosinophil lineage. In addition, some positive cells expressed neutrophil elastase, which was used as a polymorphonuclear neutrophil (PMN) marker. After birth, OSM mRNA was expressed in tissue eosinophils in nonhematopoietic organs, including small intestine, lung, and skin. Our data revealed that eosinophil progenitors and eosinophils as well as PMNs are also an important cellular source of OSM in mice.

Up-regulated phosphorylation of signal transducer and activator of transcription 3 and cyclic AMP-responsive element binding protein by peripheral inflammation in primary afferent neurons possibly through oncostatin M receptor.

Oncostatin M (OSM), a member of interleukin-6 family cytokines, contributes to the development of nociceptive sensory neurons. However, little is known about the role of OSM in dorsal root ganglia (DRGs) of adult mice after peripheral inflammation. In the present study, we showed that OSM mRNA was highly expressed in the inflamed skin during acute inflammation induced by complete Freunds adjuvant (CFA), while the expression of oncostatin M receptor (OSMR) did not change in the ipsilateral DRG. Although peripheral inflammation induced significant increases in the number of neurons with phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated p38 mitogen-activated protein kinase (p-p38) in ipsilateral DRGs, OSMR-positive neurons exhibited neither p-ERK nor p-p38. In addition, we found significant increases in the number of neurons with phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and phosphorylated cAMP-responsive element binding protein (p-CREB) in the ipsilateral DRGs. Interestingly, OSMR-positive neurons with p-STAT3 and p-CREB were significantly increased after peripheral inflammation. Thus, our results suggest that acute inflammation induce the phosphorylations of several signal molecules, including ERK, p38, cAMP-responsive element binding protein, and STAT3. Among them, the up-regulation of p-STAT3 and p-CREB may be induced possibly through OSMR.

Expression of mKirre, a mammalian homolog of Drosophila kirre, in the developing and adult mouse brain

mKirre, a mammalian homolog of the Drosophila kirre, is expressed in bone marrow stromal cells and the brain. Although mKirre has been shown to support the hematopoietic stem cells, little is known about the function of mKirre in the brain. In the present study, to gain insights into the function of mKirre, we investigated the expression pattern of mKirre gene in the developing and adult mouse brain using in situ hybridization. In the adult brain, mKirre mRNA was highly expressed in the olfactory bulb, the piriform cortex, the cochlear nucleus, and the cerebellum. At embryonic day (E) 11.5, we could observe mKirre mRNA in the differentiating zones of various regions, such as the caudate-putamen, the geniculate body, the thalamus, the amygdala, and the brainstem. Its gene expression in these regions at E11.5 also persisted to the adult, in which its expression levels were much less prominent. After birth, we could first observe high expression of mKirre mRNA in the glomerular and mitral layers of the olfactory bulb, the cortical plate of the neocortex, the cochlear nucleus, and the molecular and granule cell layers of the cerebellum. In the hippocampus, its gene expression was first observed in the dentate gyrus at postnatal day 7. The spatiotemporal expression pattern of mKirre mRNA suggests important roles of mKirre in later developmental processes, especially the synapse formation.

Subcellular localization of glucose transporter 4 in the hypothalamic arcuate nucleus of ob/ob mice under basal conditions

Glucose transporter (GLUT) 4 plays an important role in insulin-induced glucose uptake in skeletal muscle and white adipose tissue. Although GLUT4 is abundant in the hypothalamus as well as in these peripheral tissues, little is known about the role of GLUT4 in the hypothalamus. In this study, we examined the subcellular localization of GLUT4 and the activation of insulin signaling pathways in the hypothalamic arcuate nucleus of ob/ob mice under basal conditions. The expression of GLUT4 in the arcuate nucleus of ob/ob mice was higher than that in lean mice. Interestingly, GLUT4 on the plasma membrane increased significantly in neurons of the arcuate nucleus of ob/ob mice when compared to that in lean mice. Because serum insulin levels of ob/ob mice were very high, we hypothesized that insulin strongly stimulates GLUT4 translocation in the arcuate nucleus of ob/ob mice. Unexpectedly, tyrosine phosphorylation of IR and insulin receptor substrate-1 (IRS-1) was faint in the hypothalamus of lean and ob/ob mice. In addition, phosphorylation of IRS-1 at Ser307 in the hypothalamus of ob/ob mice was higher when compared to that in lean mice, suggesting that insulin signaling is impaired by phosphorylation of IRS-1 at Ser307 in the hypothalamus of ob/ob mice. However, serine phosphorylation of Akt in the arcuate nucleus of ob/ob mice increased significantly when compared to that in lean mice. Furthermore, the expression of brain-derived neurotrophic factor, an activator of PI3K-Akt pathway in neurons, increased significantly in the ventromedial hypothalamus of ob/ob mice. We discuss the possibility of novel pathways which induce the translocation of GLUT4 in the arcuate nucleus of ob/ob mice.