RESUMEN
We report the generation and characterization of transgenic mouse and zebrafish expressing green fluorescent protein (GFP) specifically in vascular endothelial cells in a relatively uniform fashion. These reporter lines exhibit fluorescent vessels in developing embryos and throughout adulthood, allowing visualization of the general vascular patterns with single cell resolution. Furthermore, we show the ability to purify endothelial cells from whole embryos and adult organs by a single step fluorescence activated cell sorting. We expect that these transgenic reporters will be useful tools for imaging vascular morphogenesis, global gene expression profile analysis of endothelial cells, and high throughput screening for vascular mutations.
Asunto(s)
Endotelio Vascular/fisiología , Proteínas Luminiscentes/genética , Proteínas Tirosina Quinasas Receptoras/genética , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Endotelio Vascular/crecimiento & desarrollo , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes , Corazón/embriología , Heterocigoto , Homocigoto , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Noqueados/embriología , Ratones Noqueados/crecimiento & desarrollo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor TIE-2 , Cola (estructura animal)/embriologíaRESUMEN
Bone morphogenetic protein (BMP) controls osteoblast proliferation and differentiation through Smad proteins. Here we show that Tob, a member of the emerging family of antiproliferative proteins, is a negative regulator of BMP/Smad signaling in osteoblasts. Mice carrying a targeted deletion of the tob gene have a greater bone mass resulting from increased numbers of osteoblasts. Orthotopic bone formation in response to BMP2 is elevated in tob-deficient mice. Overproduction of Tob represses BMP2-induced, Smad-mediated transcriptional activation. Finally, Tob associates with receptor-regulated Smads (Smad1, 5, and 8) and colocalizes with these Smads in the nuclear bodies upon BMP2 stimulation. The results indicate that Tob negatively regulates osteoblast proliferation and differentiation by suppressing the activity of the receptor-regulated Smad proteins.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Osteoblastos/fisiología , Transducción de Señal/fisiología , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta , Alelos , Animales , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 7 , Remodelación Ósea/fisiología , Diferenciación Celular/fisiología , División Celular/fisiología , Tamaño de la Célula/fisiología , Expresión Génica/fisiología , Mutación de Línea Germinal/fisiología , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/citología , Fosfoproteínas/metabolismo , Cráneo/citología , Proteínas Smad , Proteína Smad1 , Proteína Smad5 , Proteína Smad8 , Transcripción Genética/fisiologíaRESUMEN
The biological roles of estrogen-responsive finger protein (efp) in vivo were evaluated in mice carrying a loss-of-function mutation in efp by gene-targeted mutagenesis. Although efp homozygous mice were viable and fertile in both sexes, the uterus that expressed abundant estrogen receptor alpha exhibited significant underdevelopment. When the ovariectomized homozygotes were subjected to 17beta-estradiol treatment, they showed remarkably attenuated responses to estrogen, as exemplified by decreased interstitial water imbibition and retarded endometrial cell increase, at least, attributable to the lower ratio of G1 to S-phase progression in epithelial cells. These results suggest that efp is essential for the normal estrogen-induced cell proliferation and uterine swelling as one of the direct targets of estrogen receptor alpha.
Asunto(s)
Proteínas de Unión al ADN/genética , Estrógenos/metabolismo , Receptores de Estrógenos/metabolismo , Factores de Transcripción/genética , Útero/fisiología , Animales , Bromodesoxiuridina/metabolismo , Ciclo Celular/fisiología , Receptor alfa de Estrógeno , Femenino , Biblioteca de Genes , Homocigoto , Humanos , Inmunohistoquímica , Ratones , Ratones Noqueados , Modelos Biológicos , Modelos Genéticos , Fenotipo , Transducción de Señal , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Útero/anatomía & histología , Útero/crecimiento & desarrolloRESUMEN
Germ-line mutations of the human TSC2 tumor suppressor gene cause tuberous sclerosis (TSC), a disease characterized by the development of hamartomas in various organs. In the Eker rat, however, a germ-line Tsc2 mutation gives rise to renal cell carcinomas with a complete penetrance. The molecular mechanism for this phenotypic difference between man and rat is currently unknown, and the physiological function of the TSC2/Tsc2 product (tuberin) is not fully understood. To investigate these unsolved problems, we have generated a Tsc2 mutant mouse. Tsc2 heterozygous mutant (Tsc2+/-) mice developed renal carcinomas with a complete penetrance, as seen in the Eker rat, but not the angiomyolipomas characteristic of human TSC, confirming the existence of a species-specific mechanism of tumorigenesis caused by tuberin deficiency. Unexpectedly, approximately 80% of Tsc2+/- mice also developed hepatic hemangiomas that are not observed in either TSC or the Eker rat. Tsc2 homozygous (Tsc2-/-) mutants died around embryonic day 10.5, indicating an essential function for tuberin in mouse embryonic development. Some Tsc2-/- embryos exhibited an unclosed neural tube and/or thickened myocardium. The latter is associated with increased cell density that may be a reflection of loss of a growth-suppressive function of tuberin. The mouse strain described here should provide a valuable experimental model to analyze the function of tuberin and its association with tumorigenesis.
Asunto(s)
Carcinoma de Células Renales/genética , Genes Supresores de Tumor , Hemangioma/genética , Neoplasias Renales/genética , Neoplasias Hepáticas/genética , Proteínas Represoras/fisiología , Animales , Desarrollo Embrionario y Fetal/genética , Mutación de Línea Germinal , Heterocigoto , Homocigoto , Humanos , Ratones , Ratones Noqueados , Ratones Mutantes , Ratas , Proteínas Represoras/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de TumorRESUMEN
Oblique facial clefts constitute approximately 0.20% of all facial malformation cases in Japan and approximately 0.22% in other countries. In the present study, the proportion in our institute was approximately 0.21%, which is almost equal to that reported by WILSON et al. The ratio of male and female patients did not differ significantly from that in other countries.
Asunto(s)
Anomalías Craneofaciales/epidemiología , Cara/anomalías , Femenino , Humanos , Recién Nacido , Japón/epidemiología , Masculino , PrevalenciaRESUMEN
Receptor tyrosine kinases Flt-1 and Flk-1/KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1/KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice (flt-1(TK-/-)) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in flt-1(TK-/-) mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.
Asunto(s)
Desarrollo Embrionario y Fetal/fisiología , Neovascularización Fisiológica/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Animales , Secuencia de Bases , Permeabilidad Capilar/genética , Células Cultivadas , Cartilla de ADN , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/citología , Marcación de Gen , Linfocinas/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Zic genes encode zinc finger proteins, the expression of which is highly restricted to cerebellar granule cells and their precursors. These genes are homologs of the Drosophila pair-rule gene odd-paired. To clarify the role of the Zic1 gene, we have generated mice deficient in Zic1. Homozygous mice showed remarkable ataxia during postnatal development. Nearly all of the mice died within 1 month. Their cerebella were hypoplastic and missing a lobule in the anterior lobe. A bromodeoxyuridine labeling study indicated a reduction both in the proliferating cell fraction in the external germinal layer (EGL), from 14 d postcoitum, and in forward movement of the EGL. These findings suggest that Zic1 may determine the cerebellar folial pattern principally via regulation of cell proliferation in the EGL.
Asunto(s)
Cerebelo/embriología , Proteínas de Drosophila , Regulación del Desarrollo de la Expresión Génica , Ratones Mutantes Neurológicos , Dedos de Zinc/genética , Animales , Conducta Animal/fisiología , Cerebelo/anomalías , Cerebelo/citología , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Microscopía Electrónica , Mutagénesis/fisiología , Neuronas/química , Neuronas/ultraestructura , Células Madre/química , Células Madre/fisiología , Factores de Transcripción/genéticaRESUMEN
The Pebpb2 gene encodes a non-DNA binding subunit of the heterodimeric transcription factor, polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF), and is rearranged in inversion of chromosome 16 associated with human acute myeloid leukemia. To investigate its physiological function, Pebpb2 was mutated by a targeting strategy to generate a null mutant. The homozygous mutation in mice proved lethal in embryos around embryonic day 12.5, apparently due to massive hemorrhaging in the central nervous system. In addition, definitive hematopoiesis in the liver was severely impaired. The observed phenotype was indistinguishable from that reported for homozygous disruption of AML1, which encodes a DNA binding subunit of PEBP2/CBF. Thus, the results indicate that the two subunits function together as a heterodimeric PEBP2/CBF in vivo and that PEBP2/CBF plays an essential role in the development of definitive hematopoiesis.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Células Madre Hematopoyéticas/fisiología , Hígado/embriología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción Genética , Animales , Quimera , Mapeo Cromosómico , Cromosomas Humanos Par 16 , Cartilla de ADN , Desarrollo Embrionario y Fetal , Femenino , Genes Letales , Células Madre Hematopoyéticas/citología , Homocigoto , Humanos , Leucemia Mieloide/genética , Hígado/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis , Reacción en Cadena de la Polimerasa , Factor de Transcripción AP-2 , Saco Vitelino/fisiologíaRESUMEN
In the G-protein-coupled receptor superfamily, the opioid receptor subfamily is constituted of the three distinct opioid receptors (namely delta-, mu- and kappa-subtypes) and the receptor for nociceptin (also designated orphaninFQ). The members of the opioid receptor subfamily were known to mediate a variety of cellular inhibitory effects. The three opioid receptors are known to play central roles in mediating analgesia and many other physiological activities; however, the nociceptin receptor was identified recently and less is known about its physiological roles. Here we report the generation and characterization of mice lacking the nociceptin receptor. The knockout mice showed no significant differences in nociceptive threshold and locomotor activity compared with control mice, but they lost nociceptin-induced behavioral responses. These results indicate that the nociceptin system is not essential for regulation of nociception or locomotor activity. On the other hand, we found insufficient recovery of hearing ability from the adaptation to sound exposure in the mutant mice. Thus, the nociceptin system appears to participate in the regulation of the auditory system.
Asunto(s)
Audición/fisiología , Péptidos Opioides/farmacología , Dimensión del Dolor , Receptores Opioides/fisiología , Animales , Química Encefálica , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Hiperalgesia/fisiopatología , Inmunoglobulinas/sangre , Recuento de Linfocitos , Ratones , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Precursores de Proteínas/análisis , ARN Mensajero/análisis , Receptores Opioides/análisis , Receptores Opioides/genética , Proteínas Recombinantes/análisis , Médula Espinal/química , beta-Galactosidasa/análisis , Receptor de Nociceptina , NociceptinaRESUMEN
Estrogen is involved in the growth and development of female organs such as uterus and mammary gland. On the other hand, from clinical point of view, it is recently suggested that estrogen is effective to protect postmenopausal women from osteoporosis, coronary heart disease and Alzheimer disease. In order to study the molecular mechanism of estrogen action, we have identified an estrogen responsive gene, efp (estrogen-responsive finger protein), which might mediate estrogen action in various target organs at diverse stages and targeted mutagenesis of efp gene could help clarify physiologic actions of estrogen.
Asunto(s)
Estrógenos , Enfermedad de Alzheimer/prevención & control , Animales , Enfermedad Coronaria/prevención & control , Proteínas de Unión al ADN/genética , Estrógenos/farmacología , Estrógenos/fisiología , Femenino , Ratones , Mutagénesis , Especificidad de Órganos , Osteoporosis/prevención & control , Receptores de Estrógenos/metabolismo , Factores de Transcripción/genéticaRESUMEN
Many peptide hormone and neurotransmitter receptors belonging to the seven membrane-spanning G protein-coupled receptor family have been shown to transmit ligand-dependent mitogenic signals in vitro. However, the physiological roles of the mitogenic activity through G protein-coupled receptors in vivo remain to be elucidated. Here we have generated G protein-coupled cholecystokinin (CCK)-B/gastrin receptor deficient-mice by gene targeting. The homozygous mice showed a remarkable atrophy of the gastric mucosa macroscopically, even in the presence of severe hypergastrinemia. The atrophy was due to a decrease in parietal cells and chromogranin A-positive enterochromaffin-like cells expressing the H+,K(+)-ATPase and histidine decarboxylase genes, respectively. Oral administration of a proton pump inhibitor, omeprazole, which induced hypertrophy of the gastric mucosa with hypergastrinemia in wild-type littermates, did not eliminate the gastric atrophy of the homozygotes. These results clearly demonstrated that the G protein-coupled CCK-B/gastrin receptor is essential for the physiological as well as pathological proliferation of gastric mucosal cells in vivo.
Asunto(s)
Mucosa Gástrica/citología , Mucosa Gástrica/fisiología , Receptores de Colecistoquinina/fisiología , Animales , Atrofia , Células Cromafines/fisiología , Cromogranina A , Cromograninas/análisis , Cromograninas/fisiología , Sondas de ADN , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/patología , Gastrinas/metabolismo , Expresión Génica , Biblioteca Genómica , ATPasa Intercambiadora de Hidrógeno-Potásio/biosíntesis , Histidina Descarboxilasa/biosíntesis , Homocigoto , Humanos , Hipertrofia , Ratones , Ratones Noqueados , Omeprazol/farmacología , Células Parietales Gástricas/patología , Células Parietales Gástricas/fisiología , Receptor de Colecistoquinina B , Receptores de Colecistoquinina/biosíntesis , Receptores de Colecistoquinina/genética , Transducción de Señal , Sincalida/metabolismoRESUMEN
The ryanodine receptor type 3 (RyR-3) functions as a Ca2+-induced Ca2+ release (CICR) channel and is distributed in a wide variety of cell types including skeletal muscle and smooth muscle cells, neurons, and certain non-excitable cells. However, the physiological roles of RyR-3 are totally unclear. To gain an insight into the function of RyR-3 in vivo, we have generated mice lacking RyR-3 by means of the gene targeting technique. The mutant mice thus obtained showed apparently normal growth and reproduction. Although Ca2+-induced Ca2+ release from intracellular Ca2+ stores of the mutant skeletal muscle differed in Ca2+ sensitivity from that of wild-type muscle, excitation-contraction coupling of the mutant muscle seemed to be normal. Moreover, we could not find any significant disturbance in the smooth muscle and lymphocytes from the mutant mice. On the other hand, the mutant mice showed increased locomotor activity, which was about 2-fold greater than that of the control mice. These results indicate that the loss of RyR-3 causes no gross abnormalities and suggest that the lack of RyR-3-mediated Ca2+ signaling results in abnormalities of certain neurons in the central nervous system.
Asunto(s)
Canales de Calcio/fisiología , Proteínas Musculares/fisiología , Animales , Secuencia de Bases , Calcio/fisiología , División Celular , Genes , Activación de Linfocitos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Actividad Motora , Contracción Muscular , Músculo Liso Vascular/fisiología , Mapeo Restrictivo , Canal Liberador de Calcio Receptor de Rianodina , Bazo/citologíaRESUMEN
The inositol 1,4,5-trisphosphate (InsP3) receptor acts as an InsP3-gated Ca2+ release channel in a variety of cell types. Type 1 InsP3 receptor (IP3R1) is the major neuronal member of the IP3R family in the central nervous system, predominantly enriched in cerebellar Purkinje cells but also concentrated in neurons in the hippocampal CA1 region, caudate-putamen, and cerebral cortex. Here we report that most IP3R1-deficient mice generated by gene targeting die in utero, and born animals have severe ataxia and tonic or tonic-clonic seizures and die by the weaning period. An electroencephalogram showed that they suffer from epilepsy, indicating that IP3R1 is essential for proper brain function. However, observation by light microscope of the haematoxylin-eosin staining of the brain and peripheral tissues of IP3R1-deficient mice showed no abnormality, and the unique electrophysiological properties of the cerebellar Purkinje cells of IP3R1-deficient mice were not severely impaired.
Asunto(s)
Ataxia/metabolismo , Canales de Calcio/metabolismo , Epilepsia/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Ataxia/genética , Secuencia de Bases , Encéfalo/metabolismo , Canales de Calcio/deficiencia , Canales de Calcio/genética , Línea Celular , Cartilla de ADN , Electroencefalografía , Epilepsia/genética , Muerte Fetal , Marcación de Gen , Receptores de Inositol 1,4,5-Trifosfato , Ratones , Datos de Secuencia Molecular , Mutación , Células de Purkinje/metabolismo , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
We generated mice carrying a loss-of-function mutation in Brn-2, a gene encoding a nervous system specific POU transcription factor, by gene targeting in embryonic stem cells. In homozygous mutant embryos, migratory precursor cells for neurons of the paraventricular nuclei (PVN) and the supraoptic nuclei (SO) of the hypothalamus die at approximately E12.5. All homozygous mutants suffered mortality within 10 days after birth, possibly because of a complete deficiency of these neurons in the hypothalamus. Although neither developmental nor histological abnormalities were observed in heterozygous mice, the levels of expression of vasopressin and oxytocin in the hypothalamus of these animals were half these of wild-type mice. These results strongly suggest that Brn-2 plays an essential role in the determination and development of the PVN and SO neuronal lineages in the hypothalamus.
Asunto(s)
Hipotálamo/citología , Neuronas/citología , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Línea Celular , Movimiento Celular , Cartilla de ADN , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Genes Letales , Mutación de Línea Germinal , Proteínas de Homeodominio , Homocigoto , Hipotálamo/metabolismo , Ratones , Datos de Secuencia Molecular , Neuronas/metabolismo , Factores del Dominio POU , Neurohipófisis/embriología , Neurohipófisis/patología , Somatostatina/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Hepatocyte growth factor/scatter factor (HGF/SF) functions as a mitogen, motogen and morphogen for a variety of cultured cells. The genes for HGF/SF and its receptor (the c-met proto-oncogene product) are expressed in many tissues during the embryonic periods and in the adult. HGF/SF is thought to mediate a signal exchange between the mesenchyme and epithelia during mouse development. To examine the physiological role of HGF/SF, we generated mutant mice with a targeted disruption of the HGF/SF gene. Here we report that homozygous mutant embryos have severely impaired placentas with markedly reduced numbers of labyrinthine trophoblast cells, and die before birth. The growth of trophoblast cells was stimulated by HGF/SF in vitro, and the HGF/SF activity was released by allantois in primary culture of normal but not mutant embryos. These findings suggest that HGF/SF is an essential mediator of allantoic mesenchyme-trophoblastic epithelia interaction required for placental organogenesis.
Asunto(s)
Desarrollo Embrionario y Fetal/fisiología , Factor de Crecimiento de Hepatocito/fisiología , Placenta/embriología , Alantoides/metabolismo , Alantoides/patología , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Desarrollo Embrionario y Fetal/genética , Muerte Fetal , Factor de Crecimiento de Hepatocito/deficiencia , Factor de Crecimiento de Hepatocito/genética , Homocigoto , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis , Placenta/anomalías , Células Madre , Trofoblastos/patologíaRESUMEN
Contraction of skeletal muscle is triggered by the release of Ca2+ from the sarcoplasmic reticulum (SR) after depolarization of transverse tubules. The ryanodine receptor exists as a 'foot' protein in the junctional gap between the sarcoplasmic reticulum and the transverse tubule in skeletal muscle, and is proposed to function as a calcium-release channel during excitation-contraction (E-C) coupling. Previous complementary DNA-cloning studies have defined three distinct subtypes of the ryanodine receptor in mammalian tissues, namely skeletal muscle, cardiac and brain types. We report here mice with a targeted mutation in the skeletal muscle ryanodine receptor gene. Mice homozygous for the mutation die perinatally with gross abnormalities of the skeletal muscle. The contractile response to electrical stimulation under physiological conditions is totally abolished in the mutant muscle, although ryanodine receptors other than the skeletal-muscle type seem to exist because the response to caffeine is retained. Our results show that the skeletal muscle ryanodine receptor is essential for both muscular maturation and E-C coupling, and also imply that the function of the skeletal muscle ryanodine receptor during E-C coupling cannot be substituted by other subtypes of the receptor.
Asunto(s)
Canales de Calcio/fisiología , Contracción Muscular/fisiología , Proteínas Musculares/fisiología , Músculos/fisiología , Animales , Secuencia de Bases , Canales de Calcio/deficiencia , Canales de Calcio/genética , Línea Celular , Quimera , ADN , Estimulación Eléctrica , Muerte Fetal/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Musculares/deficiencia , Proteínas Musculares/genética , Músculos/fisiopatología , Músculos/ultraestructura , Mutación , Proteínas/análisis , ARN/análisis , Canal Liberador de Calcio Receptor de RianodinaRESUMEN
The tau gene encodes a protein (Tau) that is a major neuronal microtubule-associated protein localized mostly in axons. It has microtubule-binding and tubulin-polymerizing activity in vitro and is thought to make short crossbridges between axonal microtubules. Further, tau-transfected non-neuronal cells extend long axon-like processes in which microtubule bundles resembling those in axons are formed. In contrast, tau antisense oligonucleotides selectively suppress axonal elongation in cultured neurons. Thus tau is thought to be essential for neuronal cell morphogenesis, especially axonal elongation and maintenance. To test this hypothesis, we used gene targeting to produce mice lacking the tau gene. We show that the nervous system of tau-deficient mice appears to be normal immunohistologically. Furthermore, axonal elongation is not affected in cultured neurons. But in some small-calibre axons, microtubule stability is decreased and microtubule organization is significantly changed. We observed an increase in microtubule-associated protein 1A which may compensate for the functions of tau in large-calibre axons. Our results argue against the suggested role of tau in axonal elongation but confirm that it is crucial in the stabilization and organization of axonal microtubules in a certain type of axon.
Asunto(s)
Axones/ultraestructura , Microtúbulos/ultraestructura , Proteínas tau/deficiencia , Animales , Línea Celular , Clonación Molecular , Femenino , Hipocampo/ultraestructura , Masculino , Ratones , Morfogénesis/fisiología , Mutación , Neuronas/fisiología , Neuronas/ultraestructura , Proteínas tau/genética , Proteínas tau/fisiologíaRESUMEN
We constructed cell lines which can proliferate in response to IL-2 or IL-3 by introducing a wild-type and mutant forms of cDNAs encoding the human IL-2R p75 chain into an IL-3 dependent hematopoietic cell line which expresses the p55 chain of the IL-2R. We compared early events that were induced in these cells by IL-2 and IL-3. Analysis of protein tyrosine phosphorylation showed that two common protein bands, pp95 and pp90, were phosphorylated by stimulation of either IL-2 or IL-3, suggesting the possible sharing of part of a signal transduction pathway between IL-2R and IL-3R. Comparison of protein tyrosine phosphorylation profiles induced by IL-2 and IL-3 among a variety of cell lines revealed that the pp90 band is the common tyrosine phosphorylation substrate in the cell lines examined, although the general tyrosine phosphorylation pattern differed in each cell line. Mutant p75 molecules incapable of inducing tyrosine phosphorylation could bind and internalize IL-2, but could not support cell growth. We also found that swift changes of cytoskeletal protein organization are one of the early events caused by signal transduction through either IL-2R and IL-3R. Reorganization of cytoskeletal proteins seems to be associated with protein phosphorylation, as a significant portion of pp90 was found in a detergent-soluble fraction in IL-2 or IL-3 treated cells.