Disruption of neuromedin B receptor gene results in dysregulation of the pituitary-thyroid axis.

The level of thyrotropin (TSH) secretion is determined by the balance of TSH-releasing hormone (TRH) and thyroid hormones. However, neuromedin B (NB), a bombesin-like peptide, highly concentrated in the pituitary, has been postulated to be a tonic inhibitor of TSH secretion. We studied the pituitary-thyroid axis in adult male mice lacking NB receptor (NBR-KO) and their wild-type (WT) littermates. At basal state, NBR-KO mice presented serum TSH slightly higher than WT (18%, P< 0.05), normal intra-pituitary TSH content, and no significant changes in alpha and beta TSH mRNA levels. Serum thyroxine was normal but serum triiodothyronine (T3) was reduced by 24% (P< 0.01) in NBR-KO mice. Pituitaries of NBR-KO mice exhibited no alteration in prolactin mRNA expression but type I and II deiodinase mRNA levels were reduced by 53 and 42% respectively (P< 0.05), while TRH receptor mRNA levels were importantly increased (78%, P< 0.05). The TSH-releasing effect of TRH was significantly higher in NBR-KO than in WT mice (7.1-and 4.0-fold respectively), but, while WT mice presented a 27% increase in serum T3 (P< 0.05) after TRH, NBR-KO mice showed no change in serum T3 after TRH. NBR-KO mice did not respond to exogenous NB, while WT showed a 30% reduction in serum TSH. No compensatory changes in mRNA expression of NB or other bombesin-related peptides and receptors (gastrin-releasing peptide (GRP), GRP-receptor and bombesin receptor subtype-3) were found in the pituitary of NBR-KO mice. Therefore, the data suggest that NB receptor pathways are importantly involved in thyrotroph gene regulation and function, leading to a state where TSH release is facilitated especially in response to TRH, but probably with a less-bioactive TSH. Therefore, the study highlights the important role of NB as a physiological regulator of pituitary-thyroid axis function and gene expression.

Overexpression of gastrin-releasing peptide receptor induced layer disorganization in brain.

Gastrin-releasing peptide-preferring and neuromedin B-preferring receptors, members of the bombesin-like peptide receptor subfamily, are reported to regulate proliferation, migration and differentiation. Since they are expressed in developing brain, we postulated that the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors might be involved in normal brain development. Here we examined the effects of the overexpressions of the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors on chick brain development in vivo using a retrovirus. In the overexpressed exogenous gastrin-releasing peptide-preferring receptor brain, we found laminar disorganization in the telencephalon, tectum and particularly in the cerebellum with severe atrophy. Processes of the radial glial cells in the telencephalon and optic tectum, as well as the projections of the Bergmann glia in the cerebellum were distorted, which might disturb normal cell migration. Despite the atrophy of the cerebellum, densely-stained proliferating cell nuclear antigen- and phospho-histone H3-positive cells increased in number. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells also increased in the cerebellum, suggesting that the ectopically proliferating cells were subjected to apoptosis. Glial fibrillary acidic protein-positive cells also increased in the hyperpallium accessorium and in the outer layers of the tectum. We also found smaller and spindle-shaped cells which resembled undifferentiated embryonic tumor cells. On the other hand, the layer structures of the neuromedin B-preferring receptors overexpressed brain were well organized and developed, and the size of brain was generally enlarged. These results indicated that although the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors are involved in normal brain development, both receptors contribute and exert their effects differently.

Neuromedin B.

Neuromedin B (NMB) is one of the bombesin (BN)-related peptides in mammals. It was originally purified from pig spinal cords, and it has been shown to be present in central nervous system as well as in gastrointestinal tract. BN and its related peptides have various physiological effects. These include regulation of exocrine and endocrine secretions, smooth muscle contraction, feeding, blood pressure, blood glucose, body temperature and cell growth. NMB exerts its effect by binding to the cell surface receptor. A high affinity receptor, NMB receptor (NMB-R) has been identified. This is a G-protein coupled receptor with seven membrane-spanning regions. Upon agonist binding, several intracellular signaling cascades including phospholipase activation, calcium mobilization and protein kinase C (PKC) activation lead to expression of several genes, DNA synthesis or cellular effects such as secretion. Existence of NMB-R has been demonstrated in several brain regions, notably in olfactory and thalamic regions, and in gastrointestinal tracts. Recent analysis using NMB-R-deficient mice, generated by gene-targeting technique, enables to distinguish functional properties of NMB-R from GRP-R. In this review, molecular characterization, anatomical distribution and pharmacological properties of NMB and NMB-R will be presented. Moreover, physiological roles of NMB and its receptor demonstrated by the analysis of NMB-R-deficient mice will be reported. Comparison with GRP/GRP-R system will provide important information about BN-like peptide systems in mammals.

Distributions of two chicken bombesin receptors, bombesin receptor subtype-3.5 (chBRS-3.5) and gastrin-releasing peptide receptor (chGRP-R) mRNAS in the chicken telencephalon.

Bombesin (BN)-like peptide receptors are known to be essential to the regulation of not only homeostasis, including feeding behavior, but also of emotional systems in mammal. Recently, two novel BN receptors, chicken BN-like peptide receptor subtype-3.5 (chBRS-3.5) and gastrin-releasing peptide receptor (chGRP-R), have been identified. Here, we report the localizations of these receptors' mRNAs in the chick brain through development using in situ hybridization. First, chBRS-3.5 mRNA signals were found in the dorsal ventricular ridge at embryonic day (ED) 9. Strong signals were observed in the hyperpallium accessorium, nidopallium and nucleus basorostralis pallii, and moderate signals were found in the hippocampus, cortex piriformis, hyperpallium intercalatum, area temporo-parieto-occipitalis, nucleus striae terminalis lateralis, nucleus olfactorius anterior and organum septi lateralis at ED16. This wide expression in the pallium persisted during posthatch periods. Abundant expressions in the hyperpallium, nidopallium, considered to be similar to the mammalian cortex, as well as in the hippocampus, indicate participation of these molecules in the processing of sensory information, motor function, learning and memory. Telencephalic areas devoid of chBRS-3.5 signals were the entopallium, arcopallium anterius, globus pallidus, nucleus intrapeduncularis, tuberculum olfactorius, nucleus septalis lateralis, hypothalamic and thalamic areas. In contrast to chBRS-3.5, chGRP-R mRNA signals were found in the pallidum at ED5 and 9. At ED16, chGRP-R mRNA signals were localized in the medial striatum and hypothalamus. GRP-R expression in the hypothalamic region was phylogenically conserved. Thus, chBRS-3.5 mRNA signals were distributed in a broader region and were more intense than chGRP-R mRNA. Taken together, chGRP-R and chBRS-3.5 mRNA occurred in similar regions of mammals that express GRP-R. BN/GRP-immunoreactive neurons and varicosities were found mainly in the pallium, especially in the hyperpallium accessorium and nidopallium, and this distribution coincided with that of chBRS-3.5 mRNA. This result suggests that the endogenous ligands for chBRS-3.5 were likely BN-like peptides produced in the pallium.

Cloning and expression of the neuromedin B receptor and the third subtype of bombesin receptor genes in the mouse.

We cloned the genes for the mouse homologue of the neuromedin B receptor (NMB-R) and the bombesin receptor subtype 3 (BRS-3). Both receptor genes consist of three exons with well-conserved intron-exon borders. Although the NMB-R gene spans more than 10 kb, the BRS-3 gene spans only about 4 kb. Comparison of the mouse and human receptor sequences indicates 90% (NMB-R) and 85% (BRS-3) sequence homology at the amino-acid level. In the adult mouse, the NMB-R mRNA is expressed in the brain, testis, esophagus, intestine and uterus, whereas the BRS-3 mRNA is expressed predominantly in the brain. In the brain, the NMB-R gene expression is prominent in the thalamic and olfactory regions, and the BRS-3 gene is expressed particularly in the hypothalamic region. In mouse testis, the NMB-R gene expression is prominent, and the expression of BRS-3 mRNA is barely detected. In contrast, BRS-3 has been shown to be expressed in rat testis and guinea-pig uterus, therefore it is possible that a different subtype of the bombesin receptor mediates the same response in different species. Together with the mouse GRP-R gene cloned previously, cloning of the mouse NMB-R and BRS-3 genes permits comparison of function and structure of the three bombesin receptor subtypes in the mouse.

Migration of GnRH-immunoreactive neurons from the olfactory placode to the brain: a study using avian embryonic chimeras.

Previous studies suggest that gonadotropin-releasing hormone (GnRH) neurons appear in the olfactory placode and subsequently migrate into the brain during embryonic development. The aim of the present study was to obtain direct evidence for migration of GnRH neurons from the olfactory placode into the brain. Olfactory placodes from quail embryos were transplanted isotopically and isochronically, to replace the unilaterally ablated olfactory placodes of chick embryos. The chimeric embryos were allowed to develop for several days until they reached the embryonic stages when GnRH neurons are seen in the brain in normal embryos. Quail olfactory epithelia were formed in the host chick embryos. Quail olfactory nerves were also formed and reached the olfactory bulb or primordial olfactory bulb. GnRH-immunoreactive cells of quail origin revealed by a triple staining method were observed in the quail olfactory epithelium, quail olfactory nerve, chick olfactory bulb, and septo-preoptic area. These results indicate that GnRH neurons originate in the olfactory placode and migrate into the telencephalon including the septo-preoptic area. A migratory route of GnRH neurons was well documented by the use of a quail neuron-specific antibody, QN. The migratory route in the brain is discussed with special reference to the terminal nerve. A GnRH-immunoreactive neuronal group of chick origin appeared in the diencephalon of chimeric embryos. These diencephalic neurons may be of non-placodal origin. FMRFamide-immunoreactive neurons of quail origin were also found in the quail olfactory nerve and the host olfactory bulb, suggesting that FMRFamide neurons also originate in the olfactory placode and migrate into the brain.

Origin of luteinizing hormone-releasing hormone (LHRH) neurons in the chick embryo: effect of the olfactory placode ablation.

A unilateral olfactory placodectomy resulted in the absence of luteinizing hormone-releasing hormone-immunoreactive (LHRH-ir) cells in the olfactory-forebrain axis of the operated side, whereas the development of the LHRH neuronal system was not disturbed on the unoperated side. In the embryos in which a fragment of the medial olfactory epithelium was spared the damage, a small number of LHRH-ir cells were detected in the nasal region of the operated side, where the lack of the central projection of the olfactory nerve caused stagnation of LHRH-ir cells, no ir-cells being found in the brain area. These results suggest that LHRH neurons originate in the olfactory placode and then migrate into the forebrain along the olfactory nerve.

Differential effects of social isolation upon body weight, food consumption, and responsiveness to novel and social environment in bombesin receptor subtype-3 (BRS-3) deficient mice.

The effects of social isolation on body weight gain, food consumption, and responsiveness to novel and social environment were assessed in an animal model for obesity, bombesin receptor subtype-3 (BRS-3) deficient mice. In Experiment 1, body weight gain and food consumption of group- and isolation-housed wild-type and BRS-3-deficient mice were compared. In wild-type mice, group-housed animals showed greater mean body weight gain and food consumption than did the isolation-housed cohort in the early stage of the experiment, whereas in BRS-3-deficient mice, the isolation-housed mice showed greater body weight gain and food consumption than the group-housed cohort by prolonged isolation housing. In Experiment 2, isolation-housed wild-type mice exhibited increased stereotypic and vertical movements relative to group-housed subjects in a novel environment, but this effect was not observed in BRS-3-deficient mice. In Experiment 3, when social response was assessed in animals housed in isolation, BRS-3-deficient mice exhibited lower social responses than did wild-type mice. We conclude that BRS-3-deficient mice and wild-type mice are differentially affected by social isolation. These results suggest that BRS-3 expression in the CNS may affect the neural mechanisms that regulate isolation effects in wild-type animals.

Hyperresponsiveness to palatable and aversive taste stimuli in genetically obese (bombesin receptor subtype-3-deficient) mice.

Taste preference in obese mice was examined using genetically obese (bombesin receptor subtype-3: BRS-3 deficient) animals. Preference for either sodium saccharin (0.2%). sodium chloride (0.9%), citric acid (0.1%), or quinine sulfate (0.002%) solution was examined using a two-bottle test situation, and BRS-3 deficient mice not only showed a stronger preference for saccharin solution, but also a stronger aversive response to quinine solution, relative to wild-type littermates. Furthermore, a conditioned taste-aversion test measured the consumption of sodium saccharin (0.2%) and sodium chloride (0.9%) solutions after intraperitoneal injection of LiCl (0.3 M, 1 mg/kg), and BRS-3-deficient mice exhibited stronger aversion to both solutions than did control animals. In situ hybridization demonstrated that the BRS-3 gene is expressed in the parabrachial nucleus, the medial and central nuclei of the amygdala, and the hypothalamic nuclei such as paraventricular nucleus, all of which are known to be involved in taste perception. These results suggest that expression of the BRS-3 gene in these nuclei is important for the modulation of taste preference, as well as the development of obesity.

Successful xenogeneic transplantation in embryos: induction of tolerance by extrathymic chick tissue grafted into quail.

In previous experiments, we have demonstrated that limb buds engrafted during embryonic life at E4, between MHC-mismatched chick embryos, are not only tolerated after birth, but induce in the recipient a state of split tolerance toward cells expressing the donor MHC haplotype: donor's skin grafts are permanently tolerated while a proliferative response of host's T cells is generated in MLR by donor-type blood cells. If the same experiment is performed, using quail embryo as a donor and chick as a recipient, acute rejection of the quail limb starts during the first two weeks after birth, thus suggesting that the peripheral type of tolerance induced in these experiments can be obtained only in allogeneic but not in xenogeneic combinations. We report here the unexpected result that when a chick limb bud is grafted into a quail at E4, it is tolerated and, like allogeneic grafts in chickens, induces adult skin-graft tolerance without modifying the MLR response. Similar results were obtained with grafts from another closely related species of bird, the guinea fowl from the Phasianidae family. In contrast, xenogeneic combinations involving more distant species (chick and quail as recipients and duck, an Anatidae, as donor) resulted in strong and early rejection from both recipients. As a whole, quails exhibit a greater ability than the chick to become tolerant to antigens presented peripherally from early developmental stages. In adult quails, however, skin grafts performed in either direction (i.e., quail to chick or the reverse) are rejected according to a similar temporal pattern. Moreover, lymphocytes of both species are able to respond equally well to quail or chick IL-2. Several hypotheses are envisaged to account for these observations. It seems likely that this type of tolerance is directly related to antigenic load because the load in chick to quail wing chimeras is larger than that in quail to chick chimeras. This view is supported by the protracted delay in graft rejection observed when two quail wing buds instead of one are grafted into chickens.

TCR repertoire in early fetal mouse thymus.

We investigated the rearrangement and expression of TCR genes in mouse fetal thymus organ culture, a system that avoids subsequent entry of hematopoietic precursor cells. The first observable rearranged TCR gene was homogeneous V gamma 2-J gamma 2, detectable as early as fetal day 11 (d11) in the thymic primordia. The productive TCR was homogeneous V gamma 5-J gamma 1, first detectable in d13 thymocytes, followed by adult-type TCR gamma (V gamma 4 and V gamma 7). Sequence analysis of TCR revealed five types of V-J junctional sequences. In the very early stage, a homogeneous V-J junction is generated via a short homology sequence in the coding region (Type I), while a short homology sequence in the P-nucleotide rather than the coding region is used in the following stage (Type II). In the later embryonic stages, diverse V-J junctions are generated by well-known mechanisms, such as P-nucleotide (Type III), N-region insertion (Type IV) or trimming of the coding ends (Type V). These findings suggest that the generation of homogeneous TCR gamma (V gamma 2 and V gamma 5) in the early fetal stages is due to the intrinsic rearrangement mechanisms and is in stage specific manner.

Control of the limb bud outgrowth in quail-chick chimera.

It is well known that the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) play a major role in growth and patterning of the limb. But a mechanism underlying species-specific growth of the limb has not yet been fully elucidated. To investigate the role of AER and ZPA in limb size control, we constructed quail-chick limb chimeras. When we grafted a whole forelimb bud from one species to another, the size of the developed grafted limb was comparable to the limb of the donor species. Moreover, we demonstrated that neither the interspecific substitution of the posterior half region of the limb bud containing the ZPA nor the exchange of the ectodermal component of the limb involving the AER could alter the species-specific size of the limb. These results indicate that the factors affecting the size of the limb are already involved in the mesodermal component of the limb bud at stage 20 of chick embryo. Thus, the mesoderm dictates limb specificity including size.

Functional properties of two bombesin-like peptide receptors revealed by the analysis of mice lacking neuromedin B receptor.

The neuromedin B-preferring receptor (NMB-R) is one of the members of the bombesin (BN)-like peptide receptor subfamily in mammals. Previously, we have generated and characterized mice with targeted disruption of the two other BN-like peptide receptors, bombesin receptor subtype-3 (BRS-3) and gastrin-releasing peptide-preferring receptor (GRP-R). Here we describe the generation and analysis of NMB-R-deficient mice to investigate how NMB-R differs from BRS-3 and GRP-R. Compensation for NMB-R deficiency by overexpression of GRP-R and/or BRS-3 was not detected. Although the hypothermic effect of NMB was reduced by 50% in NMB-R-deficient mice, the effect of GRP infusion was comparable to the wild-type mice. In contrast, fundic smooth muscle contraction on stimulation with NMB or GRP was normal in NMB-R-deficient mice. Administration of GRP but not NMB suppressed glucose intake in both normal and NMB-R-deficient mice. These results suggest that the NMB-R has an essential role in thermoregulation, but not for smooth muscle contraction of the fundus or for the suppression of feeding behavior. In addition, the behavioral phenotypes of GRP-R-deficient mice were not observed in NMB-R-deficient mice. These data show that the functions of NMB-R and GRP-R are distinct, with only partial overlap.

Mice lacking bombesin receptor subtype-3 develop metabolic defects and obesity.

Mammalian bombesin-like peptides are widely distributed in the central nervous system as well as in the gastrointestinal tract, where they modulate smooth-muscle contraction, exocrine and endocrine processes, metabolism and behaviour. They bind to G-protein-coupled receptors on the cell surface to elicit their effects. Bombesin-like peptide receptors cloned so far include, gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype-3 (BRS-3). However, despite the molecular characterization of BRS-3, determination of its function has been difficult as a result of its low affinity for bombesin and its lack of an identified natural ligand. We have generated BRS-3-deficient mice in an attempt to determine the in vivo function of the receptor. Mice lacking functional BRS-3 developed a mild obesity, associated with hypertension and impairment of glucose metabolism. They also exhibited reduced metabolic rate, increased feeding efficiency and subsequent hyperphagia. Our data suggest that BRS-3 is required for the regulation of endocrine processes and metabolism responsible for energy balance and adiposity. BRS-3-deficient mice provide a useful new model for the investigation of human obesity and associated diseases.

Generation and characterization of mice lacking gastrin-releasing peptide receptor.

Gastrin-releasing peptide (GRP) is a mammalian bombesin-like peptide which is widely distributed in the central nervous system as well as in the gastrointestinal tract. GRP binds to its high affinity receptor (GRPR) to elicit a wide spectrum of biological effects on behavior, digestion, and metabolism. To define the in vivo function of GRPR, we generated GRPR null mutant mice by gene targeting. The intracerebroventricular administration of GRP caused hypothermia in wild-type mice, but not in mutant mice. The GRPR deficient mice showed significantly increased locomotor activity during the dark period, and social responses scored by sniffing, mounting, and approaching behaviors against an intruder. Aggressive scores such as fighting and biting were not altered in the mutant mice. These phenotypes were observed in mice generated from two independent ES cell clones and backcrossed to a C57BL/6J background. The GRPR deficient mice should be useful for studying the bombesin system in vivo.