RESUMEN
Multiple endocrine neoplasia type 2B (MEN2B) is an autosomal dominant syndrome characterized by the development of medullary thyroid carcinoma, pheochromocytomas, musculoskeletal anomalies and mucosal ganglioneuromas. MEN2B is caused by a specific mutation (Met918-->Thr) in the RET receptor tyrosine kinase. Different mutations of RET lead to other conditions including MEN2A, familial medullary thyroid carcinoma and intestinal aganglionosis (Hirschsprung disease). Transgenic mice were created using the dopamine beta-hydroxylase promoter to direct expression of RET(MEN2B) in the developing sympathetic and enteric nervous systems and the adrenal medulla. DbetaH-RET(MEN2B) transgenic mice developed benign neuroglial tumors, histologically identical to human ganglioneuromas, in their sympathetic nervous systems and adrenal glands. The enteric nervous system was not affected. The neoplasms in DbetaH-RET(MEN2B) mice were similar to benign neuroglial tumors induced in transgenic mice by activated Ras expression under control of the same promoter. Levels of phosphorylated MAP kinase were not increased in the RET(MEN2B)-induced neurolglial proliferations, suggesting that alternative pathways may play a role in the pathogenesis of these lesions. Transgenic mice with the highest levels of DbetaH-RET(MEN2B) expression, unexpectedly developed renal malformations analogous to those reported with loss of function mutations in the Ret gene.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Proteínas de Drosophila , Ganglioneuroma/genética , Regulación de la Expresión Génica , Riñón/anomalías , Neoplasia Endocrina Múltiple Tipo 2a/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Sistema Nervioso Simpático , Glándulas Suprarrenales/inervación , Glándulas Suprarrenales/patología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Humanos , Hiperplasia/genética , Ratones , Ratones Transgénicos , Neoplasias del Sistema Nervioso Periférico/genética , Proteínas Proto-Oncogénicas c-ret , Sistema Nervioso Simpático/patologíaRESUMEN
Metallothionein-III (MT-III), a brain-specific member of the metallothionein family of metal-binding proteins, is abundant in glutamatergic neurons that release zinc from their synaptic terminals, such as hippocampal pyramidal neurons and dentate granule cells. MT-III may be an important regulator of zinc in the nervous system, and its absence has been implicated in the development of Alzheimer's disease. However, the roles of MT-III in brain physiology and pathophysiology have not been elucidated. Mice lacking MT-III because of targeted gene inactivation were generated to evaluate the neurobiological significance of MT-III. MT-III-deficient mice had decreased concentrations of zinc in several brain regions, including hippocampus, but the pool of histochemically reactive zinc was not disturbed. Mutant mice exhibited normal spatial learning in the Morris water maze and were not sensitive to systemic zinc or cadmium exposure. No neuropathology or behavioral deficits were detected in 2-year-old MT-III-deficient mice, but the age-related increase in glial fibrillary acidic protein expression was more pronounced in mutant brain. MT-III-deficient mice were more susceptible to seizures induced by kainic acid and subsequently exhibited greater neuron injury in the CA3 field of hippocampus. Conversely, transgenic mice containing elevated levels of MT-III were more resistant to CA3 neuron injury induced by seizures. These observations suggest a potential role for MT-III in zinc regulation during neural stimulation.
Asunto(s)
Genes , Ratones Noqueados/genética , Proteínas del Tejido Nervioso/genética , Envejecimiento/metabolismo , Animales , Conducta Animal/fisiología , Encéfalo/metabolismo , Encéfalo/patología , Cadmio/antagonistas & inhibidores , Susceptibilidad a Enfermedades , Proteína Ácida Fibrilar de la Glía/metabolismo , Aprendizaje/fisiología , Memoria/fisiología , Metalotioneína 3 , Metales/farmacología , Ratones , Proteínas del Tejido Nervioso/fisiología , Neuronas/efectos de los fármacos , Convulsiones/patología , Zinc/antagonistas & inhibidores , Zinc/metabolismoRESUMEN
Sympathetic neurons, enteric neurons and adrenal chromaffin cells all derive from the neural crest. During development these cells migrate, proliferate, survive and differentiate in a highly controlled fashion influenced by local signals encountered during their migration. Aberrations of these processes are responsible for a variety of developmental defects and malignancies. Many of the environmental signals influencing these precursor cells activate receptor tyrosine kinases that can signal, at least in part, via Ras pathways. To assess the extent to which Ras can alter neuroblast cell number and fate in vivo, we expressed activated H-Ras in transgenic mice using the dopamine-beta-hydroxylase promoter, which directs expression to these cells prior to and after their differentiation. Ganglioneuromas and occasional neuroblastomas formed in the adrenal gland and preaortic sympathetic ganglia. Curiously, neurons of the superior cervical ganglia and the gut were largely unaffected despite demonstrated expression of activated Ras. The sensitivity of preaortic sympathetic neurons and adrenal chromaffin cells to the effects of oncogenes such as Ras may explain the predilection of neuroblastomas in humans to these sites. The ability to analyse neuroblastoma development in these mice may shed light on the molecular basis of certain types of human neuroblastoma.
Asunto(s)
Transformación Celular Neoplásica/patología , Neuronas/patología , Proteínas ras/metabolismo , Glándulas Suprarrenales/patología , Médula Suprarrenal/inervación , Médula Suprarrenal/patología , Aneuploidia , Animales , Diferenciación Celular , Transformación Celular Neoplásica/metabolismo , Aberraciones Cromosómicas/genética , Trastornos de los Cromosomas , Dopamina beta-Hidroxilasa/genética , Femenino , Ganglios Simpáticos/patología , Genes myc , Humanos , Hiperplasia , Cariotipificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Transgénicos , Neuritas/patología , Neuroblastoma/genética , Neuronas/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/genética , Proteínas ras/genéticaRESUMEN
Metallothionein (MT)-bound zinc accumulates when animals are exposed to excess zinc and is depleted under conditions of zinc deficiency, suggesting that MT serves as a means of sequestering excess zinc as well as a zinc reservoir that can be utilized when zinc is deficient. To examine the importance of MT for these processes, mice with null alleles of both MT I and MT II genes were created and the zinc concentration and histological appearance of multiple organs assessed. At birth, the hepatic zinc concentration of these MT-null mice was lower than that of wild-type controls (0.27 +/- 0.02 vs. 0.65 +/- 0.11 micromol zinc/g tissue, P < 0.05). During the next 3 wk of suckling zinc-replete (95 micrograms zinc/g diet) dams, the hepatic zinc concentration of controls fell to 0.42 +/- 0.04 micromol/g but was unchanged in the MT-null mice (0.28 +/- 0.04 micromol/g). The most prominent histological anomaly observed at 3 wk of age was the presence of swollen Bowman's capsules in the kidneys of MT-null mice. When nursing MT-null dams were fed a severely zinc-deficient (1.5 microg/g) diet, kidney development in the MT-null pups was retarded as indicated by the retention of the nephrogenic zone and incomplete tubule development. We suggest that the lack of a hepatic reservoir of zinc jeopardizes the developing kidney in the MT-null mice. In addition to being more sensitive to dietary zinc restriction, MT-null mice are more sensitive to zinc toxicity. When adult mice were challenged with a ramping dose of zinc up to a total of 3700 micromol zinc/kg body weight, MT-null mice had a greater incidence of pancreatic acinar cell degeneration compared with control mice despite accumulating less zinc (2.72 +/- 0.46 vs. 1.23 +/- 0.52 micromol zinc/g pancreas, control and MT-null, respectively, P < 0.05). The results of these experiments suggest that MT I and MT II can protect against both zinc deficiency and zinc toxicity.
Asunto(s)
Riñón/metabolismo , Hígado/metabolismo , Metalotioneína/fisiología , Zinc/deficiencia , Zinc/toxicidad , Animales , Animales Recién Nacidos , Peso Corporal/efectos de los fármacos , Huesos/metabolismo , Dieta , Genotipo , Riñón/patología , Masculino , Metalotioneína/biosíntesis , Ratones , Ratones Noqueados , Páncreas/efectos de los fármacos , Páncreas/patologíaRESUMEN
We cloned and sequenced the mouse phenylethanolamine N-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5' flanking sequences from the cloned mouse PNMT gene can direct expression of the Escherichia coli beta-galactosidase (lacZ) gene to predicted regions of the adrenal, eye and brain in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT8 kb-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Feniletanolamina N-Metiltransferasa/genética , Regiones Promotoras Genéticas/genética , Médula Suprarrenal/química , Médula Suprarrenal/citología , Animales , Química Encefálica , Clonación Molecular , Toxina Diftérica/genética , Desarrollo Embrionario y Fetal , Femenino , Genes Reporteros/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Especificidad de Órganos , Proteínas Recombinantes de Fusión/biosíntesis , Retina/química , Retina/citología , Análisis de Secuencia de ADNRESUMEN
MT-III, a brain-specific member of the metallothionein gene family, binds zinc and may facilitate the storage of zinc in neurons. The distribution of MT-III mRNA within the adult brain was determined by solution and in situ hybridization and compared to that of MT-I mRNA. MT-III mRNA is particularly abundant within the cerebral cortex, hippocampus, amygdala, and nuclei at base of the cerebellum. Transgenic mice generated using 11.5 kb of the mouse MT-III 5' flanking region fused to the E. coli lacZ gene express beta-galactosidase in many of the same regions identified by in situ hybridization. MT-III mRNA was present in readily identifiable neurons within the olfactory bulb, hippocampus, and cerebellum, and beta-galactosidase activity was localized to neurons throughout the brain, but not to glia, as determined by costaining with X-Gal and neural- and glia-specific antibodies. There is marked correspondence between the neurons that are rich in MT-III mRNA and those neurons that store zinc in their terminal vesicles. MT-III is found complexed with zinc in vivo and its expression in cultured cells leads to the intracellular accumulation of zinc and enhanced histochemical detection of zinc. These results are discussed in light of the possibility that MT-III may participate in the utilization of zinc as a neuromodulator.
Asunto(s)
Metalotioneína/metabolismo , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Zinc/metabolismo , Animales , Elementos sin Sentido (Genética)/análisis , Secuencia de Bases , Encéfalo/embriología , Química Encefálica , ADN/análisis , Expresión Génica , Metalotioneína/genética , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Neuronas/química , ARN Mensajero/análisis , Valores de ReferenciaRESUMEN
A new member of the metallothionein (MT) gene family was discovered that lies about 20 kb 5' of the MT-III gene in both mouse and human. The MT-IV proteins are highly conserved in both species and have a glutamate insertion at position 5 relative to the classical MT-I and MT-II proteins. Murine MT-IV mRNA appears to be expressed exclusively in stratified squamous epithelia associated with oral epithelia, esophagus, upper stomach, tail, footpads, and neonatal skin. The MT derived from tongue epithelium contains both zinc and copper. Many of these epithelia develop parakeratosis during zinc deficiency in the rat. In situ hybridization reveals intense labeling of MT-IV mRNA in the differentiating spinous layer of cornified epithelia, whereas MT-I is expressed predominantly in the basal, proliferative layer; thus, there is a switch in MT isoform synthesis during differentiation of these epithelia. We suggest that MT-IV plays a special role in regulating zinc metabolism during the differentiation of stratified epithelia.
Asunto(s)
Células Epiteliales , Metalotioneína/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Clonación Molecular , Epitelio/metabolismo , Expresión Génica , Humanos , Hibridación in Situ , Metalotioneína/genética , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Zinc/metabolismoRESUMEN
Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone, is a glycoprotein normally secreted by the Sertoli cells of the fetal and adult testis and by granulosa cells of the postnatal ovary. The production of MIS in the male fetus brings about the regression of the Müllerian ducts, the anlagen of the uterus, oviducts, and upper vagina. In addition, purified MIS induces the formation of seminiferous cord-like structures in fetal rat ovaries cultured in vitro, suggesting that MIS may influence testicular differentiation. We have produced transgenic mice chronically expressing human MIS under the control of the mouse metallothionein-1 promoter to investigate its role during sexual development. In females, chronic expression led to the inhibition of Müllerian duct differentiation, resulting in a blind vagina and no uterus or oviducts. At birth the ovaries had fewer germ cells than normal; during the next two weeks germ cells were lost and the somatic cells became organized into structures resembling seminiferous tubules. Apparently, these structures degenerate as they are undetectable in adult females. The majority of transgenic males developed normally. But in two lines with the highest levels of MIS expression, some males showed feminization of the external genitalia, impairment of Wolffian duct development, and undescended testes. These results suggest that MIS has several distinct roles in mammalian sexual development.