Tomoko Hisaoka

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.

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.

Site-specific subtypes of macrophages recruited after peripheral nerve injury.

After partial ligation of mouse sciatic nerve, the subtypes of macrophages were examined in the injured nerve and dorsal root ganglia (DRGs). Many M1 macrophages, which were inducible nitric oxide synthase (iNOS)-positive and arginase-1 (Arg-1)-negative, and neutrophils infiltrated the injured nerve. In contrast, almost all macrophages infiltrating the ipsilateral side of DRGs after the nerve injury were iNOS−/Arg-1+, M2 type. The infiltration of M1 and M2 macrophages was first observed in the injured nerve and ipsilateral DRGs on days 1 and 2, respectively. In addition, the macrophage infiltration preceded the activation of microglia in the ipsilateral dorsal horn of spinal cord. Thus, infiltrating macrophages after peripheral nerve injury may play unique roles dependent on the location in the development of neuropathic pain.

Expression of a member of tumor necrosis factor receptor superfamily, TROY, in the developing mouse brain

TROY is a recently identified member of the tumor necrosis factor (TNF) receptor superfamily. We investigated the expression pattern of TROY mRNA in the developing central nervous system by the in situ hybridization technique. TROY mRNA was strongly expressed in the ventricular zone and the subventricular zone, which contain neuronal and glial precursors during mouse embryogenesis. Its spatial and temporal expression patterns suggest that TROY plays some important roles in neurogenesis of embryonic stages.

Expression of a member of tumor necrosis factor receptor superfamily, TROY, in the developing olfactory system.

TROY is a recently identified member of the tumor necrosis factor (TNF) receptor superfamily. We have previously reported that TROY induces the activation of nuclear factor κB via TNF receptor‐associated factor 2, 5, and 6, and is strongly expressed in the developing central nervous system, including the olfactory bulb. In this study, we investigated the detailed cellular characterization of TROY‐expressing cells in the developing olfactory system of mice using in situ hybridization and immunohistochemistry. Both mRNA and protein of TROY were first detected in the olfactory nerve layer (ONL) of the olfactory bulb at embryonic day 13.5. During late embryogenesis, TROY expression was most intense in the inner ONL (ONL‐i). In the postnatal olfactory bulb, TROY‐expressing cells were also detected in the glomerular layer (GL), in addition to the ONL‐i. The double‐immunofluorescence method demonstrated that TROY was expressed in olfactory ensheathing cells (OECs) of the ONL‐i, which were positive for neuropeptide Y (NPY), but neither S‐100 nor p75 low‐affinity nerve growth factor receptor. Some TROY‐expressing cells in the ONL‐i were observed with the astrocyte‐like phenotype (GFAP+/NPY−). In addition, TROY was also detected in GFAP+ glial cells of the GL. Thus, TROY was expressed in some specific subsets of glial cells in the olfactory bulb, including OECs, suggesting that TROY may play some roles in the developing and adult olfactory system.

The Absence of Interleukin-6 Enhanced Arsenite-Induced Renal Injury by Promoting Autophagy of Tubular Epithelial Cells with Aberrant Extracellular Signal-Regulated Kinase Activation

Sodium arsenite (NaAs)-induced autophagic cell death (ACD) of a mouse renal tubular epithelial cell line (mProx24), which expresses enhanced levels of interleukin-6 (IL-6), was reduced by the suppression of autophagy by 3-methyladenine or Atg7 knockdown. The inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) signal pathway by anti-IL-6 antibody or a Jak2 inhibitor (AG490) exaggerated ACD of mProx24 cells after NaAs challenge, attenuating STAT3 activation and reciprocally enhancing extracellular signal-regulated kinase (ERK) phosphorylation. In contrast, an ERK inhibitor, PD98059, reduced NaAs-induced ACD in mProx24 cells. Subcutaneous injection of NaAs (12.5 mg/kg) into BALB/c (wild-type) mice enhanced intrarenal expression of IL-6, mainly produced by tubular cells, and caused severe renal injury characterized by hemorrhages, acute tubular necrosis, cast formation, and brush border disappearance, with increases in serum urea nitrogen (blood urea nitrogen) and creatinine levels. In addition, IL-6-deficient (IL-6(-/-)) mice exhibited exaggerated histopathological changes with higher blood urea nitrogen and creatinine levels. Moreover, in IL-6(-/-) mice treated with NaAs, ACD in renal tubular cells was significantly augmented, along with diminished STAT3 activation and reciprocal enhancement of ERK signaling, compared with wild-type mice. Finally, the administration of exogenous IL-6 into wild-type mice significantly reduced NaAs-induced ACD along with diminished ERK activation and eventually alleviated acute renal dysfunction. Thus, IL-6/STAT3 signal pathway could inhibit ERK activation, a crucial step for ACD, eventually attenuating NaAs-induced renal dysfunction.

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.

Characterization of Foxp2-expressing cells in the developing spinal cord.

Two members of winged-helix/forkhead transcription factors, Foxp1 and Foxp2, are expressed in the developing and adult CNS, including the striatum, cerebral cortex, and thalamus. In a previous study, we have demonstrated that Foxp1 is expressed in a subpopulation of V1 interneurons in addition to motor neurons of the spinal cord during mouse embryogenesis. However, the detailed expression pattern of Foxp2 and its relationship with Foxp1 in the developing spinal cord remains to be elucidated. To shed light on the potential roles of Foxp1 and Foxp2 in the developing spinal cord, we characterized Foxp2-expressing cells during mouse embryogenesis. At embryonic day (E) 11.0, Foxp2-expressing cells were first observed in the ventral spinal cord, which were Pax6(-), p27(+), and neuron-specific class III beta-tubulin(+) postmitotic neurons. Between E13.5 and E15.5, high expression of Foxp2 was observed in both medial and lateral parts of the ventral spinal cord. Double-immunofluorescence staining for Foxp2 with some homeodomain transcription factors revealed that Foxp2-expressing neurons were Pax2(+), En1(+), Evx1(-), Chx10(-), Gata3(-), and Lhx3(-) V1 interneurons in the intermediate zone throughout the ventral spinal cord, indicating that Foxp2-expressing neurons were also V1 interneurons with the same phenotypes as Foxp1-expressing interneurons. In addition, neither Foxp1 nor Foxp2 was expressed in ventral calbindin(+) Renshaw cells. However, Foxp2 did not colocalize with Foxp1 in interneurons of the ventral spinal cord. These findings suggest that Foxp1 and Foxp2 are expressed in the distinct subsets of V1 interneurons that belong to non-Renshaw cells in the ventral spinal cord during embryogenesis. Thus, Foxp1 and Foxp2 may be involved in the determination of the cell type identities during late embryogenesis: the classes of neurotransmitters and the functional subtypes of non-Renshaw cells, such as Ia and Ib inhibitory interneurons.

Expression of mKirre in the developing sensory pathways: Its close apposition to nephrin-expressing cells

mKirre is a novel member of the immunoglobulin superfamily, which is abundant in the developing and adult brain. In the present study, we showed mKirre gene expression in mouse sensory organs during development using in situ hybridization and immunohistochemistry. At embryonic day (E) 11.5, E15.5, and E17.5, we first detected signals for mKirre mRNA in the developing cochleae, retinae, and olfactory neuroepithelia, respectively. After birth, strong signals were observed in these sensory organs. In addition, at this stage, we found its expression in trigeminal ganglion neurons and neuronal populations forming sensory pathways in the olfactory bulb, midbrain, and pons. Furthermore, double-immunofluorescence staining revealed that nephrin-immunoreactivity was overlapping to mKirre-expressing cells in the developing sensory organs. These results suggest that mKirre may be involved in the establishment of the pathway from sensory organs to the brain not only in a homophilic manner but also with its heterophilic interaction to nephrin.

Characterization of TROY/TNFRSF19/TAJ-expressing cells in the adult mouse forebrain

A member of the tumor necrosis factor receptor superfamily (TNFRSF), TROY/TNFRSF19/TAJ, is highly expressed in the brain of adult mice. Northern blot analysis using mRNA taken from regions of the adult CNS showed the expression of TROY in all regions examined, including the olfactory bulb, cerebral cortex, striatum, and hippocampus. In situ hybridization and immunohistochemistry revealed that TROY mRNA and protein were strongly expressed in the rostral migratory stream (RMS) and subventricular zone (SVZ) of adult mice. In the adult SVZ, some glial fibrillary acidic protein (GFAP)-positive cells (type B cells) are thought to be multipotent neural stem cells. These type B cells divide slowly and generate epidermal growth factor receptor (EGFR)-positive transit-amplifying precursor cells (type C cells) in the presence of epidermal growth factor (EGF). Type C cells give rise to neuron-specific class III beta-tubulin (TuJ1)-positive neuroblasts (type A cells) that migrate to the olfactory bulb along the RMS. TROY-expressing cells were GFAP-positive, EGFR-positive, and TuJ1-negative in the adult SVZ. From these findings, TROY appears to be expressed in type B and type C cells, but not in type A cells, which was supported by immunoelectron microscopy. In addition, TROY was expressed in GFAP-positive astrocytes of the various regions, such as the cerebral cortex, striatum, and hippocampus. Thus, TROY was expressed in uncommitted precursor cells and astroglial lineage cells, suggesting that TROY plays some roles in the regulation of gliogenesis in the adult CNS.