The effect of laminin-1 on enteric neural crest-derived cell migration in the Hirschsprung's disease mouse model.

BACKGROUND/AIM: Laminin-1 regulates neurite outgrowth in various neuronal cells. We have previously demonstrated that laminin-1 promotes enteric neural crest-derived cell (ENCC) migration by using Sox10-VENUS transgenic mice, in which ENCCs are labeled with a green fluorescent protein, Venus. Mice lacking the endothelin-B receptor gene, Ednrb -/- mice, are widely used as a model for Hirschsprung's disease (HD). The aim of this study was to investigate the effects of laminin-1on ENCC migration in Sox10-VENUS+/Ednrb -/- mice, a newly created HD mice model. METHODS: Fetal guts were dissected on embryonic day 12.5 (E12.5). Specimens were incubated either with, or without laminin-1 for 24 h and images were taken under a stereoscopic microscope. The length from the stomach to the wavefront of ENCC migration (L-E) and the total length of the gut (L-G) were measured. Changes in the ratio of L-E to L-G (L-E/L-G) after 24 h were calculated. RESULTS: On E12.5, the wavefront of ENCC migration in the HD gut samples was located in the midgut, whereas the wavefront of ENCC in Sox10-VENUS+/Ednrb +/+ (WT) samples had reached the hindgut. After 24 h, L-E/L-G had increased by 1.49%, from 34.97 to 36.46%, in HD gut and had increased by 1.07%, from 48.08 to 49.15%, in HD with laminin-1, suggesting there was no positive effect of laminin-1 administration on ENCC migration in HD. CONCLUSIONS: Our results suggest that laminin-1 does not have a positive effect on ENCC migration in HD mice on E12.5, in contrast to the phenomenon seen in normal mice gut specimens, where laminin-1 promotes ENCC migration during the same period. This suggests that there is an impairment in the interaction between ENCC and extracellular environmental factors, which are required for normal development of the enteric nervous system, resulting in an aganglionic colon in HD.

Not single but periodic injections of synovial mesenchymal stem cells maintain viable cells in knees and inhibit osteoarthritis progression in rats.

OBJECTIVE: We investigated the effects of single or repetitive intra-articular injections of synovial mesenchymal stem cells (MSCs) on a rat osteoarthritis (OA) model, and elucidated the behaviors and underlying mechanisms of the stem cells after the injection. DESIGN: One week after the transection of the anterior cruciate ligament (ACL) of wild type Lewis rats, one million synovial MSCs were injected into the knee joint every week. Cartilage degeneration was evaluated with safranin-o staining after the first injection. To analyze cell kinetics or MSC properties, luciferase, LacZ, and GFP expressing synovial MSCs were used. To confirm the role of MSCs, species-specific microarray and PCR analyses were performed using human synovial MSCs. RESULTS: Histological analysis for femoral and tibial cartilage showed that a single injection was ineffective but weekly injections had significant chondroprotective effects for 12 weeks. Histological and flow-cytometric analyses of LacZ and GFP expressing synovial MSCs revealed that injected MSCs migrated mainly into the synovium and most of them retained their undifferentiated MSC properties though the migrated cells rapidly decreased. In vivo imaging analysis revealed that MSCs maintained in knees while weekly injection. Species-specific microarray and PCR analyses showed that the human mRNAs on day 1 for 21 genes increased over 50-fold, and increased the expressions of PRG-4, BMP-2, and BMP-6 genes encoding chondroprotective proteins, and TSG-6 encoding an anti-inflammatory one. CONCLUSION: Not single but periodic injections of synovial MSCs maintained viable cells without losing their MSC properties in knees and inhibited osteoarthritis (OA) progression by secretion of trophic factors.

Purified Human Dental Pulp Stem Cells Promote Osteogenic Regeneration.

Human dental pulp stem/progenitor cells (hDPSCs) are attractive candidates for regenerative therapy because they can be easily expanded to generate colony-forming unit-fibroblasts (CFU-Fs) on plastic and the large cell numbers required for transplantation. However, isolation based on adherence to plastic inevitably changes the surface marker expression and biological properties of the cells. Consequently, little is currently known about the original phenotypes of tissue precursor cells that give rise to plastic-adherent CFU-Fs. To better understand the in vivo functions and translational therapeutic potential of hDPSCs and other stem cells, selective cell markers must be identified in the progenitor cells. Here, we identified a dental pulp tissue-specific cell population based on the expression profiles of 2 cell-surface markers LNGFR (CD271) and THY-1 (CD90). Prospectively isolated, dental pulp-derived LNGFR(Low+)THY-1(High+) cells represent a highly enriched population of clonogenic cells--notably, the isolated cells exhibited long-term proliferation and multilineage differentiation potential in vitro. The cells also expressed known mesenchymal cell markers and promoted new bone formation to heal critical-size calvarial defects in vivo. These findings suggest that LNGFR(Low+)THY-1(High+) dental pulp-derived cells provide an excellent source of material for bone regenerative strategies.

The existence of experience and frequency and severity of related concerns of ethical issues in nursing practice encountered in organ transplantation.

OBJECTIVES: The purpose of this study was to elucidate the existence of the experience of and the frequency and severity of related concerns of distressful ethical situations encountered by nursing professionals in organ transplantation. METHODS: An anonymous self-administered questionnaire was mailed to 569 nurses in 79 facilities that performed organ transplantation with living or brain-dead donors who provided approval for this study. The questionnaire, developed according to the Likert method, was composed of 12 items referring to the basic attributes of nursing professionals based on the results of previous studies and the scientific literature, as well as 27 items referring to the presence or absence and the frequency and severity of concerns regarding ethical situations. The data were analyzed using descriptive statistics. RESULTS: The questionnaire was distributed to 569 nursing professionals working in 79 facilities that had provided consent for study participation. Responses were obtained from 218 participants (recovery rate: 38.3%). Among the 3 highest-ranking items, those in the first and second positions in terms of the presence or absence and the frequency of worries were the same as those in the second and third positions in terms of the severity of concerns. In addition, the 3 lower-ranking items also were the same. Among the ethical situations encountered by nursing professionals, the ones most often experienced that caused the most concern were the following: "I have questioned whether it was better for the recipient, who could not do self-care after the transplant, to undergo transplantation", and "I have felt that a recipient decided to receive a transplant without considering the importance of posttransplant self-management when making a decision about transplant surgery." CONCLUSIONS: The results indicate that most of the ethical issues related to organ transplantation in nursing practice were experienced because recipients, their families, and donors could not foresee the various problems that might occur after transplantation.

Self-management of infection control behavior of adult recipients of living-donor liver transplantation within 5 years after transplantation.

This study determines the present condition of self-management of infection control behavior of adult recipients who underwent living-donor liver transplantation (LDLT). The design was a qualitative study using a semistructured interview. The subjects were recipients who underwent LDLT at Kyoto University Hospital within 5 years to March 2011 and gave their consents to participate in this study. The subjects were 10 recipients (4 male and 6 female), and their average age was 56.7 years. Of 502 sentences about self-management behavior extracted from the verbatim records of all subjects, 61 sentences were about infection control behavior. Cluster analysis was used to classify these sentences into 5 groups: basic preventive behavior, application preventive behavior, active preventive behavior, change of preventive behavior depending on physical condition, and establishment of preventive behavior.

Differential cellular localization of antioxidant enzymes in the trigeminal ganglion.

Because of its high oxygen demands, neural tissue is predisposed to oxidative stress. Here, our aim was to clarify the cellular localization of antioxidant enzymes in the trigeminal ganglion. We found that the transcriptional factor Sox10 is localized exclusively in satellite glial cells (SGCs) in the adult trigeminal ganglion. The use of transgenic mice that express the fluorescent protein Venus under the Sox10 promoter enabled us to distinguish between neurons and SGCs. Although both superoxide dismutases 1 and 2 were present in the neurons, only superoxide dismutase 1 was identified in SGCs. The enzymes relevant to hydrogen peroxide degradation displayed differential cellular localization, such that neurons were endowed with glutathione peroxidase 1 and thioredoxin 2, and catalase and thioredoxin 2 were present in SGCs. Our immunohistochemical finding showed that only SGCs were labeled by the oxidative damage marker 8-hydroxy-2'-deoxyguanosine, which indicates that the antioxidant systems of SGCs were less potent. The transient receptor potential vanilloid subfamily member 1 (TRPV1), the capsaicin receptor, is implicated in inflammatory hyperalgesia, and we demonstrated that topical capsaicin application causes short-lasting mechanical hyperalgesia in the face. Our cell-based assay revealed that TRPV1 agonist stimulation in the presence of TRPV1 overexpression caused reactive oxygen species-mediated caspase-3 activation. Moreover, capsaicin induced the cellular demise of primary TRPV1-positive trigeminal ganglion neurons in a dose-dependent manner, and this effect was inhibited by a free radical scavenger and a pancaspase inhibitor. This study delineates the localization of antioxidative stress-related enzymes in the trigeminal ganglion and reveals the importance of the pivotal role of reactive oxygen species in the TRPV1-mediated caspase-dependent cell death of trigeminal ganglion neurons. Therapeutic measures for antioxidative stress should be taken to prevent damage to trigeminal primary sensory neurons in inflammatory pain disorders.

The use of megavoltage cone-beam CT to complement CT for target definition in pelvic radiotherapy in the presence of hip replacement.

In Europe and the USA combined, over half a million people had a hip joint replaced in 2005, contributing to the increasing number of radiotherapy patients with metallic hip prostheses. The treatment plan for external beam radiation therapy is based on the delineation of the anatomy in the planning CT scan. When implanted objects of high atomic number (Z) material are present, however, severe image artefacts are generated in conventional CT, strongly hindering the ability to delineate some organs. This is particularly the case for the planning of prostate patients with hip prostheses. This short communication presents the use of a new imaging modality, megavoltage cone-beam CT, to complement the regular CT for target definition of prostate cancer treatment of patients with hip replacements.

Visualization of microglia in living tissues using Iba1-EGFP transgenic mice.

Microglia are thought to play important roles not only in repairing injured tissue but in regulating neuronal activity, and visualizing the cells is very useful as a means of further investigating the function of microglia in vivo. We previously cloned the ionized calcium-binding adaptor molecule 1 (Iba1) gene, which is expressed selectively in microglia/microphages. To generate new transgenic mice to visualize microglia with enhanced green fluorescent protein (EGFP), we here constructed a plasmid carrying EGFP cDNA under control of the Iba1 promoter. This construct was injected into C57B/6 mouse zygotes, and the Iba1-EGFP transgenic line was developed. Fluorescent in-situ hybridization analysis revealed that the Iba1-EGFP transgene was located on chromosome 11D. No obvious defects were observed during development or in adulthood, and the EGFP fluorescence remained invariant over the course of at least four generations. Judging from the immunoreactivity with anti-Iba1 antibody, all EGFP-positive cells in the adult brain were ramified microglia. In the developing transgenic embryos, EGFP signals were detected as early as embryonic Day 10.5. The most prominent EGFP signals were found in forebrain, spinal cord, eye, foreleg, yolk sac, liver, and vessel walls. At postnatal Day 6, clear EGFP signals were observed in the supraventricular corpus callosum, known as "fountain of microglia", where ameboid microglia migrate into the brain parenchyma and mature into ramified microglia. Iba1-EGFP transgenic mice thus permit observation of living microglia under a fluorescence microscope and provide a useful tool for studying the function of microglia in vivo.

Distribution of the galectin-1 mRNA in the rat nervous system: its transient upregulation in rat facial motor neurons after facial nerve axotomy.

Galectin-1 is a member of the animal lectin family that displays conserved consensus sequences and similar carbohydrate binding specificities. Recent analyses revealed that galectin-1 plays an important role in the process of nerve regeneration. We analyzed the topological expression of galectin-1 mRNA in adult rat nervous system. Galectin-1 mRNA was predominantly observed in the cell bodies of neurons such as oculomotor nucleus (III), trochlear nucleus (IV), trigeminal motor nucleus (V), abducens nucleus (VI), facial nucleus (VII), hypoglossal nucleus (XII), red nucleus, and locus ceruleus. Neurons in pineal gland and dorsal root ganglia expressed galectin-1 mRNA. We next tested whether the axotomy of facial nerve altered the expression of galectin-1 mRNA in motor neurons. In the adult rats, the axotomy of facial nerve induced transient upregulation of galectin-1 mRNA around 6 h after axotomy. These results indicate that galectin-1 may play roles in the early event of the nerve injury and regeneration through the transient change of its expression level.

Molecular characterization of mammalian homologues of class C Vps proteins that interact with syntaxin-7.

Vesicle-mediated protein sorting plays an important role in segregation of intracellular molecules into distinct organelles. Extensive genetic studies using yeast have identified more than 40 vacuolar protein sorting (VPS) genes involved in vesicle transport to vacuoles. However, their mammalian counterparts are not fully elucidated. In this study, we identified two human homologues of yeast Class C VPS genes, human VPS11 (hVPS11) and human VPS18 (hVPS18). We also characterized the subcellular localization and interactions of the protein products not only from these genes but also from the other mammalian Class C VPS homologue genes, hVPS16 and rVPS33a. The protein products of hVPS11 (hVps11) and hVPS18 (hVps18) were ubiquitously expressed in peripheral tissues, suggesting that they have a fundamental role in cellular function. Indirect immunofluorescence microscopy revealed that the mammalian Class C Vps proteins are predominantly associated with late endosomes/lysosomes. Immunoprecipitation and gel filtration studies showed that the mammalian Class C Vps proteins constitute a large hetero-oligomeric complex that interacts with syntaxin-7. These results indicate that like their yeast counterparts, mammalian Class C Vps proteins mediate vesicle trafficking steps in the endosome/lysosome pathway.

Three-dimensional intensity-modulated radiotherapy in the treatment of nasopharyngeal carcinoma: the University of California-San Francisco experience.

PURPOSE: To review our experience with three-dimensional intensity-modulated radiotherapy (IMRT) in the treatment of nasopharyngeal carcinoma. METHODS AND MATERIALS: We reviewed the records of 35 patients who underwent 3D IMRT for nasopharyngeal carcinoma at the University of California-San Francisco between April 1995 and March 1998. According to the 1997 American Joint Committee on Cancer staging classification, 4 (12%) patients had Stage I disease, 6 (17%) had Stage II, 11 (32%) had Stage III, and 14 (40%) had Stage IV disease. IMRT of the primary tumor was delivered using one of the following three techniques: (1) manually cut partial transmission blocks, (2) computer-controlled autosequencing static multileaf collimator (MLC), and (3) Peacock system using a dynamic multivane intensity-modulating collimator (MIMiC). A forward 3D treatment-planning system was used for the first two methods, and an inverse treatment planning system was used for the third method. The neck was irradiated with a conventional technique using lateral opposed fields to the upper neck and an anterior field to the lower neck and supraclavicular fossae. The prescribed dose was 65-70 Gy to the gross tumor volume (GTV) and positive neck nodes, 60 Gy to the clinical target volume (CTV), and 50-60 Gy to the clinically negative neck. Eleven (32%) patients had fractionated high-dose-rate intracavitary brachytherapy boost to the primary tumor 1-2 weeks following external beam radiotherapy. Thirty-two (91%) patients also received cisplatin during, and cisplatin and 5-fluorouracil after, radiotherapy. Acute and late normal tissue effects were graded according to the Radiation Therapy Oncology Group (RTOG) radiation morbidity scoring criteria. Local-regional progression-free, distant metastasis-free survival and overall survival were estimated using the Kaplan-Meier method. RESULTS: With a median follow-up of 21.8 months (range, 5-49 months), the local-regional progression-free rate was 100%. The 4-year overall survival was 94%, and the distant metastasis-free rate was 57%. The worst acute toxicity was Grade 2 in 16 (46%) patients, Grade 3 in 18 (51%) patients and Grade 4 in 1 (3%) patient. The worst late toxicity was Grade 1 in 15 (43%), Grade 2 in 13 (37%), and Grade 3 in 5 (14%) patients. Only 1 patient had a transient Grade 4 soft-tissue necrosis. At 24 months after treatment, 50% of the evaluated patients had Grade 0, 50% had Grade 1, and none had Grade 2 xerostomia. Analysis of the dose-volume histograms (DVHs) showed that the average maximum, mean, and minimum dose delivered were 79.5 Gy, 75.8 Gy, and 56.5 Gy to the GTV, and 78.9 Gy, 71.2 Gy, and 45.4 Gy to the CTV, respectively. An average of only 3% of the GTV and 2% of the CTV received less than 95% of the prescribed dose. The average dose to 5% of the brain stem, optic chiasm, and right and left optic nerves was 48.3 Gy, 23.9 Gy, 15.0 Gy, and 14.9 Gy, respectively. The average dose to 1 cc of the cervical spinal cord was 41.7 Gy. The doses delivered were within the tolerance of these critical normal structures. The average dose to 50% of the right and left parotids, pituitary, right and left T-M joints, and ears was 43. 2 Gy, 41.0 Gy, 46.3 Gy, 60.5 Gy, 58.3 Gy, 52.0 Gy, and 52.2 Gy, respectively. CONCLUSION: 3D intensity-modulated radiotherapy provided improved target volume coverage and increased dose to the gross tumor with significant sparing of the salivary glands and other critical normal structures. Local-regional control rate with combined IMRT and chemotherapy was excellent, although distant metastasis remained unabated.

Comparison of treatment plans involving intensity-modulated radiotherapy for nasopharyngeal carcinoma.

PURPOSE: To compare intensity-modulated radiotherapy (IMRT) treatment plans with conventional treatment plans for a case of locally advanced nasopharyngeal carcinoma. METHODS AND MATERIALS: The study case was planned using two types of IMRT techniques, as well as a three-dimensional conformal radiotherapy technique (3D-CRT), and a traditional treatment method using bilateral opposing fields. These four plans were compared with respect to dose conformality, dose-volume histogram (DVH), dose to the sensitive normal tissue structures, and ease of treatment delivery. RESULTS: The planned dose distributions were more conformal to the tumor target volume in the IMRT plans than those in the conventional plans. With similar dose coverage of the clinical target volume (CTV), defined as delivery of minimum of 60 Gy to >/= 95% of CTV, the IMRT plans achieved better sensitive normal tissue structure sparing, while concomitantly delivering a minimum dose of 68 Gy to >/= 95% of the gross tumor volume (GTV) at a higher dose per fraction. CONCLUSIONS: Compared to conventional techniques, IMRT techniques provide improved tumor target coverage with significantly better sparing of sensitive normal tissue structures in the treatment of locally advanced nasopharyngeal carcinoma. With improvement of the delivery efficiency, IMRT should provide the optimal treatment for all nasopharyngeal carcinoma. Further studies are needed to establish the true clinical advantage of this new modality.

Dose optimization for the treatment of anaplastic thyroid carcinoma: a comparison of treatment planning techniques.

PURPOSE: To evaluate and compare dose optimization for the treatment of anaplastic thyroid carcinoma using a 3D conformal plan, and two 3D intensity-modulated inverse plans. METHODS AND MATERIALS: After patient immobilization using an alpha cradle and head-mask system, a postoperative CT scan was obtained to delineate the gross tumor volume (GTV), the clinical tumor volume (CTV), and adjacent critical structures. Treatment plans were generated using UM-Plan (University of Michigan), PeacockPlan and Corvus (NOMOS Corporation, Sewickley, PA). Isodoses were displayed in the sagittal, coronal, and multiple axial planes, and dose-volume histograms (DVH) were generated for the GTV, CTV, and critical normal tissues. Treatment times were estimated to compare the practicality of delivering each plan in a busy radiotherapy department. RESULTS: All three treatment planning systems were able to deliver a minimum dose of 60 Gy to the GTV while keeping the maximum spinal cord dose at or below 45 Gy. However, there were differences in the doses delivered to 50% and 5% of the cord, the minimum CTV dose, and the overall treatment time. The PeacockPlan best spared the uninvolved tissues of the posterior neck, and provided the lowest dose to the cord without compromising the CTV. CONCLUSIONS: Inverse treatment planning provides superior dose optimization for the treatment of anaplastic thyroid carcinoma. The radiobiologic impact of intensity modulation for this tumor should be further tested clinically.

Live fluorescence imaging reveals early recruitment of emerin, LBR, RanBP2, and Nup153 to reforming functional nuclear envelopes.

We determined the times when the nuclear membrane, nuclear pore complex (NPC) components, and nuclear import function were recovered during telophase in living HeLa cells. Simultaneous observation of fluorescently-labeled NLS-bearing proteins, lamin B receptor (LBR)-GFP, and Hoechst33342-stained chromosomes revealed that nuclear membranes reassembled around chromosomes by 5 minutes after the onset of anaphase (early telophase) whereas nuclear import function was recovered later, at 8 minutes. GFP-tagged emerin also accumulated on chromosomes 5 minutes after the onset of anaphase. Interestingly, emerin and LBR initially accumulated at distinct, separate locations, but then became uniform 8 minutes after the onset of anaphase, concurrent with the recovery of nuclear import function. We further determined the timing of NPC assembly by immunofluorescence staining of cells fixed at precise times after the onset of anaphase. Taken together, these results showed that emerin, LBR, and several NPC components (RanBP2, Nup153, p62), but not Tpr, reconstitute around chromosomes very early in telophase prior to the recovery of nuclear import activity.

Expression of human GFR alpha-1 (GDNF receptor) at the neuromuscular junction and myelinated nerves.

Motor neurons have been known to require a wide variety of neurotrophic factors for their survival. As one of the target-derived trophic factors, glial cell line-derived neurotrophic factor (GDNF) has been shown to exert its effects on motor neurons via a receptor complex including GDNF receptor alpha 1 (GFR alpha-1). Immunoreactivity of GFR alpha-1 was observed at myelinated peripheral nerves and neuromuscular junction (NMJ) of human skeletal muscles. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that mRNA of GFR alpha-1 existed in the ventral horn of human spinal cord, but not in the skeletal muscles. The results suggested that GFR alpha-1 might play a key role for uptake and internalization of GDNF at the human NMJ.

Prominent expression of glial cell line-derived neurotrophic factor in human skeletal muscle.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to exert neurotrophic effects on motor neurons as well as mesencephalic dopaminergic neurons. Because GDNF promotes survival of motor neurons in vivo and in vitro and rescues motor neurons from naturally occurring cell death, the potential use of GDNF for treatment of motor neuron diseases has been a major focus of recent research. The expression of GDNF in humans, however, has not been fully examined. In the present study, we examined the expression of GDNF in adult human muscle by Northern blot, reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemical analyses to address physiological roles of GDNF in humans. Northern blot analysis demonstrated high expression of GDNF mRNA in human skeletal muscle when compared to that of mouse. Intense GDNF immunoreactivity was observed in the vicinity of plasma membranes of skeletal muscle, particularly at neuromuscular junctions. GDNF immunoreactivity was also observed within the axons and surrounding Schwann cells of peripheral nerves. However, RT-PCR detected expression of GDNF mRNA only in skeletal muscle, and not within the anterior horn cells of human spinal cord. These results suggest that GDNF is produced by skeletal muscle and taken up at the nerve terminals for retrograde transport by axons. Thus, GDNF in human skeletal muscle may be involved in promoting motor neuron survival as a target-derived neurotrophic factor.

Up-regulation of glial cell line-derived neurotrophic factor (GDNF) expression in regenerating muscle fibers in neuromuscular diseases.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to exert a target-derived trophic factor for motor neurons. Immunohistochemical analyses revealed that expression of GDNF in regeneration muscle fibers was up-regulated in polymyositis (PM) and Duchenne type muscular dystrophy (DMD). Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that the full length GDNF was up-regulated in PM and DMD muscle; normal muscle exhibited mostly truncated GDNF. The results indicate that the GDNF expression is regulated in regeneration of human skeletal muscle.

Labeling neural cells using adenoviral gene transfer of membrane-targeted GFP.

We describe an experimental system to visualize the soma and processes of mammalian neurons and glia in living and fixed preparations by using a recombinant adenovirus vector to transfer the jellyfish green fluorescent protein (GFP) into postmitotic neural cells both in vitro and in vivo. We have introduced several modifications of GFP that enhance its fluorescence intensity in mammalian axons and dendrites. This method should be useful for studying the dynamic processes of cell migration and the development of neuronal connections, as well as for analyzing the function of exogenous genes introduced into cells using the adenovirus vector.

Distributions of the mRNAs for L-2-amino-4-phosphonobutyrate-sensitive metabotropic glutamate receptors, mGluR4 and mGluR7, in the rat brain.

The distribution of mRNAs for metabotropic glutamate receptors, mGluR4 and mGluR7, which are highly sensitive for L-2-amino-4-phosphonobutyrate (L-AP4), was examined in the central nervous system of the rat by in situ hybridization. In general, the hybridization signals of mGluR7 mRNA were more widely distributed than those of mGluR4 mRNA, and differential expression of mGluR4 mRNA and mGluR7 mRNA was clearly indicated in some brain regions. Intense or moderate expression of mGluR4 mRNA was detected in the granule cells of the olfactory bulb and cerebellum, whereas no significant expression of mGluR7 mRNA was found in these cells. In other neurons or regions where mGluR7 mRNA was intensely or moderately expressed, no significant expression of mGluR4 mRNA was observed. Such were the mitral and tufted cells of the olfactory bulb; anterior olfactory nucleus; neocortical regions; cingulate cortex; retrosplenial cortex; piriform cortex; perirhinal cortex; CA1; CA3; granule cells of the dentate gyrus; superficial layers of the subicular cortex; deep layers of the entorhinal, parasubicular, and presubicular cortices; ventral part of the lateral septal nucleus; septohippocampal nucleus; triangular septal nucleus; nuclei of the diagonal band; bed nucleus of the stria terminalis; ventral pallidum; claustrum; amygdaloid nuclei other than the intercalated nuclei; preoptic region; hypothalamic nuclei other than the medial mammillary nucleus; ventral lateral geniculate nucleus; locus coeruleus; Purkinje cells; many nuclei of the lower brainstem other than the superior colliculus, periaqueductal gray, interpeduncular nucleus, pontine nuclei, and dorsal cochlear nucleus; and dorsal horn of the spinal cord. Both mGluR4 mRNA and mGluR7 mRNA were moderately or intensely expressed in the olfactory tubercle, superficial layers of the entorhinal cortex, CA4, septofimbrial nucleus, intercalated nuclei of the amygdala, medial mammillary nucleus, many thalamic nuclei, and pontine nuclei. Intense expression of both mGluR4 mRNA and mGluR7 mRNA was further detected in the trigeminal ganglion and dorsal root ganglia, whereas no significant expression of them was found in the pterygopalatine ganglion and superior cervical ganglion. The results indicate differential roles of the L-AP4-sensitive metabotropic glutamate receptors in the glutamatergic nervous system.

MATH-2, a mammalian helix-loop-helix factor structurally related to the product of Drosophila proneural gene atonal, is specifically expressed in the nervous system.

In Drosophila, multiple helix-loop-helix (HLH) factors play an essential role in neural development. Mammalian homologues of such Drosophila HLH factors have been recently characterized and provide useful information for the analysis of the mechanisms of mammalian neurogenesis. We report here the molecular characterization of a novel mouse HLH factor, designated MATH-2, that has a structural homology to the product of Drosophila proneural gene atonal. MATH-2 consists of 337 amino acid residues and contains an atonal-related basic HLH domain. However, outside of this domain, there is no significant sequence similarity between MATH-2 and Atonal. MATH-2 expression occurs by embryonic day 11.5 (E11.5), and is first detected in the wall of brain vesicles as well as in the spinal cord. It is expressed in the cortical plate and the mantle layer throughout the developing central nervous system but not in the ventricular zone. By E13.5, the expression becomes more prominent in the cortical plate of the cerebrum but decreases in the other regions. In the adult, the cerebrum produces a high level of MATH-2 RNA but other neural tissues produce only low levels. MATH-2 RNA is not detected in non-neural tissues, indicating that MATH-2 expression is specific to the nervous system. The gel mobility-shift analysis shows that MATH-2 can interact with several E-box sequences in collaboration with E47, a ubiquitously expressed HLH factor. These results raise the possibility that MATH-2 may be a trans-acting factor involved in the development and maintenance of the mammalian nervous system.