RESUMEN
The HMG-domain transcription factor Sox10 is essential for the development of various neural crest derived lineages including glia and neurons of the peripheral nervous system (PNS). Within the PNS the most striking defect is the complete absence of glial differentiation whereas neurogenesis seemed initially normal. A degeneration of motoneurons and sensory neurons occurred later in development. The mechanism that leads to the dramatic effects on the neural crest derived cell lineages in the dorsal root ganglia (DRG), however, has not been examined up to now. Here, we provide a detailed analysis of proliferation and apoptosis in the DRG during the time of their generation and lineage segregation (between E 9.5 and E 11.5). We show that both increased apoptosis as well as decreased proliferation of neural crest cells contribute to the observed hypomorphism.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/fisiología , Sistema Nervioso Periférico/embriología , Apoptosis , Diferenciación Celular , División Celular , Linaje de la Célula , Proteínas de Unión al ADN/genética , Marcadores Genéticos , Heterocigoto , Proteínas del Grupo de Alta Movilidad/genética , Homocigoto , Inmunohistoquímica , Hibridación in Situ , Operón Lac , Microscopía Fluorescente , Cresta Neural/citología , Estructura Terciaria de Proteína , Factores de Transcripción SOXE , Factores de Tiempo , Factores de TranscripciónRESUMEN
Co-conservation of sequence and function is an important principle during evolution. As a consequence, sequence-related genes often have similar functions in evolutionarily distant species. Enter the 'glial cells missing' (gcm) genes. They code for a small family of novel transcription factors that share DNA-binding properties and domain structure. However, no evolutionarily conserved function is apparent as yet. The prototypical gcm from Drosophila dominates nervous system development as a fate switch and master regulator of gliogenesis, whereas mammalian gcm genes have roles in placental morphogenesis and development of the parathyroid gland. Apparently, structure and function sometimes can go separate ways.
Asunto(s)
Crecimiento/genética , Neuropéptidos/genética , Transactivadores/genética , Animales , Secuencia Conservada , ADN/metabolismo , Proteínas de Unión al ADN , Proteínas de Drosophila , Drosophila melanogaster , Humanos , Sistema Nervioso/crecimiento & desarrollo , Glándulas Paratiroides/crecimiento & desarrollo , Placentación , Conformación Proteica , Factores de TranscripciónRESUMEN
The molecular mechanisms that determine glial cell fate in the vertebrate nervous system have not been elucidated. Peripheral glial cells differentiate from pluripotent neural crest cells. We show here that the transcription factor Sox10 is a key regulator in differentiation of peripheral glial cells. In mice that carry a spontaneous or a targeted mutation of Sox10, neuronal cells form in dorsal root ganglia, but Schwann cells or satellite cells are not generated. At later developmental stages, this lack of peripheral glial cells results in a severe degeneration of sensory and motor neurons. Moreover, we show that Sox10 controls expression of ErbB3 in neural crest cells. ErbB3 encodes a Neuregulin receptor, and down-regulation of ErbB3 accounts for many changes in development of neural crest cells observed in Sox10 mutant mice. Sox10 also has functions not mediated by ErbB3, for instance in the melanocyte lineage. Phenotypes observed in heterozygous mice that carry a targeted Sox10 null allele reproduce those observed in heterozygous Sox10(Dom) mice. Haploinsufficiency of Sox10 can thus cause pigmentation and megacolon defects, which are also observed in Sox10(Dom)/+ mice and in patients with Waardenburg-Hirschsprung disease caused by heterozygous SOX10 mutations.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Cresta Neural/citología , Neuroglía/citología , Animales , Diferenciación Celular , Quimera , Ganglios Espinales/embriología , Heterocigoto , Homocigoto , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuroblastoma , Neuroglía/fisiología , Ratas , Receptor ErbB-3/genética , Factores de Transcripción SOXE , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , beta-Galactosidasa/genéticaRESUMEN
Myelinating glia express high levels of a unique set of genes which code for structural proteins of the myelin sheath. Few transcription factors have so far been implicated in the regulation of any myelin gene. Here we show that the protein zero (P(0)) gene, a myelin gene exclusively expressed in the Schwann cell lineage of the peripheral nervous system, is controlled in its expression by the high-mobility-group domain protein Sox10 both in tissue culture and in vivo. Induction of wild-type Sox10, but not of other transcription factors or Sox10 mutants, strongly increased endogenous P(0) expression in tissue culture. This activation was mediated by the P(0) promoter, which was stimulated by Sox10 in transient transfections. Detailed analyses revealed the involvement of a proximal and a distal promoter region. The distal region functioned only in conjunction with the proximal one and contained a single Sox consensus binding site, which accounted for most of its activity. In contrast, the proximal region mediated Sox10 responsiveness on its own. It contained multiple binding sites for Sox proteins, with two high-affinity sites being the most significant. P(0) expression also depended on Sox10 in vivo, as evident from the analysis of Schwann cell precursors in mouse embryos with Sox10 mutation at day 12.5 of embryogenesis. To our knowledge this is the most conclusive link to date between a glial transcription factor and cell-specific activation of myelin gene expression.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas del Grupo de Alta Movilidad/fisiología , Proteína P0 de la Mielina/genética , Animales , Secuencia de Bases , Western Blotting , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Regulación de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Hibridación in Situ , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXE , Nervios Espinales/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
The GCM family of transcription factors consists of Drosophila melanogaster GCM, an important regulator of gliogenesis in the fly, and its two mammalian homologs, GCMa and GCMb. To clarify the function of these mammalian homologs, we deleted GCMa in mice. Genetic ablation of murine GCMa (mGCMa) is embryonic lethal, with mice dying between 9.5 and 10 days postcoitum. At the time of death, no abnormalities were apparent in the embryo proper. Nervous system development, in particular, was not impaired, as might have been expected in analogy to Drosophila GCM. Instead, placental failure was the cause of death. In agreement with the selective expression of mGCMa in labyrinthine trophoblasts, mutant placentas did not develop a functional labyrinth layer, which is necessary for nutrient and gas exchange between maternal and fetal blood. Only a few fetal blood vessels entered the placenta, and these failed to thrive and branch normally. Labyrinthine trophoblasts did not differentiate. All other layers of the placenta, including spongiotrophoblast and giant cell layer, formed normally. Our results indicate that mGCMa plays a critical role in trophoblast differentiation and the signal transduction processes required for normal vascularization of the placenta.
Asunto(s)
Desarrollo Embrionario y Fetal/genética , Neuropéptidos/genética , Placenta/fisiopatología , Transactivadores/genética , Animales , Femenino , Marcación de Gen/métodos , Genotipo , Heterocigoto , Histocitoquímica , Hibridación in Situ , Operón Lac , Ratones , Ratones Noqueados , Neuropéptidos/metabolismo , Placenta/embriología , Embarazo , Transactivadores/metabolismoRESUMEN
The ErbB2 tyrosine kinase functions as coreceptor for the neuregulin receptors ErbB3 and ErbB4 and can participate in signaling of EGF receptor (ErbB1), interleukin receptor gp130, and G-protein coupled receptors. ErbB2(-/-) mice die at midgestation because of heart malformation. Here, we report a genetic rescue of their heart development by myocardial expression of erbB2 cDNA that allows survival of the mutants to birth. In rescued erbB2 mutants, Schwann cells are lacking. Motoneurons form and can project to muscle, but nerves are poorly fasciculated and disorganized. Neuromuscular junctions form, as reflected in clustering of AChR and postsynaptic expression of the genes encoding the alpha-AChR, AChE, epsilon-AChR, and the RI subunit of the cAMP protein kinase. However, a severe loss of motoneurons on cervical and lumbar, but not on thoracic levels occurs. Our results define the roles of Schwann cells during motoneuron and synapse development, and reveal different survival requirements for distinct motoneuron populations.
Asunto(s)
Corazón/embriología , Sistema Nervioso Periférico/embriología , Receptor ErbB-2/fisiología , Factores de Transcripción , Proteínas de Xenopus , Alelos , Animales , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Ratones , Ratones Mutantes , Neuronas Motoras , Mutación , Cresta Neural , Unión Neuromuscular , Sistema Nervioso Periférico/anomalías , Receptor ErbB-2/genética , Células de Schwann , SinapsisRESUMEN
Neuregulins (NDF, heregulin, GGF ARIA, or SMDF) are EGF-like growth and differentiation factors that signal through tyrosine kinase receptors of the ErbB family. Here, we report a novel phenotype in mice with targeted mutations in the erbB2, erbB3, or neuregulin-1 genes. These three mutations cause a severe hypoplasia of the primary sympathetic ganglion chain. We provide evidence that migration of neural crest cells to the mesenchyme lateral of the dorsal aorta, in which they differentiate into sympathetic neurons, depends on neuregulin-1 and its receptors. Neuregulin-1 is expressed at the origin of neural crest cells. Moreover, a tight link between neuregulin-1 expression, the migratory path, and the target site of sympathogenic neural crest cells is observed. Sympathetic ganglia synthesize catecholamines in the embryo and the adult. Accordingly, catecholamine levels in mutant embryos are severely decreased, and we suggest that the lack of catecholamines contributes to the embryonal lethality of the erbB3 mutant mice. Thus, neuregulin-1, erbB2, and erbB3 are required for the formation of the sympathetic nervous system; the block in development observed in mutant mice is caused by a lack of neural crest precursor cells in the anlage of the primary sympathetic ganglion chain. Together with previous observations, these findings establish the neuregulin signaling system as a key regulator in the development of neural crest cells.
Asunto(s)
Antineoplásicos/farmacología , Receptores ErbB/fisiología , Glicoproteínas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Receptor ErbB-2/fisiología , Sistema Nervioso Simpático/crecimiento & desarrollo , Animales , Antineoplásicos/metabolismo , Aorta/química , Aorta/embriología , Aorta/fisiología , Movimiento Celular/fisiología , Embrión de Mamíferos/química , Embrión de Mamíferos/metabolismo , Receptores ErbB/genética , Ganglios Espinales/química , Ganglios Espinales/metabolismo , Expresión Génica/genética , Genes/genética , Genes erbB-2/genética , Glicoproteínas/genética , Ligandos , Mesodermo/química , Mesodermo/citología , Mesodermo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Mutación/genética , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/farmacología , Cresta Neural/química , Cresta Neural/citología , Cresta Neural/fisiología , Neurregulinas , Fenotipo , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-2/genética , Receptor ErbB-3 , Transducción de Señal , Sistema Nervioso Simpático/embriología , Factores de TiempoRESUMEN
Neuregulins and their specific receptors, members of the ErbB family of tyrosine kinases, have been implicated in the control of growth and development of Schwann cells, specialized cells that wrap around nerve axons to provide electrical insulation. Here we use gene targeting to generate mice that lack ErbB3, a high-affinity neuregulin receptor. Homozygous erbB3 mutant embryos lack Schwann-cell precursors and Schwann cells that accompany peripheral axons of sensory and motor neurons. The initial development of motor neurons and sensory neurons of dorsal root ganglia occurs as it should, but at later stages most motor neurons (79%) and sensory neurons in dorsal root ganglia (82%) undergo cell death in erbB3 mutant embryos. Degeneration of the peripheral nervous system in erbB3 mutant pups is thus much more severe than the cell death in mice that lack neurotrophins or neurotrophin receptors. We also show that ErbB3 functions in a cell-autonomous way during the development of Schwann cells, but not in the survival of sensory or motor neurons. Our results indicate that sensory and motor neurons require factors for their survival that are provided by developing Schwann cells.
Asunto(s)
Receptores ErbB/fisiología , Enfermedades del Sistema Nervioso/embriología , Proteínas Proto-Oncogénicas/fisiología , Animales , Receptores ErbB/genética , Eliminación de Gen , Marcación de Gen , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/patología , Neuronas Motoras/fisiología , Sistema Nervioso/embriología , Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/patología , Neuronas Aferentes/patología , Neuronas Aferentes/fisiología , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-3 , Células de Schwann/patología , Células de Schwann/fisiología , Transducción de SeñalRESUMEN
The c-ros gene was originally identified in mutant form as an oncogene. The proto-oncogene encodes a tyrosine kinase receptor that is expressed in a small number of epithelial cell types, including those of the epididymis. Targeted mutations of c-ros in the mouse reveal an essential role of the gene in male fertility. Male c-ros -/- animals do not reproduce, whereas the fertility of female animals is not affected. We demonstrate that c-ros is not required in a cell autonomous manner for male germ cell development or function. The gene, therefore, does not affect sperm generation or function in a direct manner. The primary defect in the mutant animals was located in the epididymis, showing that c-ros controls appropriate development of the epithelia, particularly regionalization and terminal differentiation. The epididymal defect does not interfere with production or storage of sperm but, rather, with sperm maturation and the ability of sperm to fertilize in vivo. Interestingly, sperm isolated from c-ros -/- animals can fertilize in vitro. Our results highlight the essential role of the epididymis in male fertility and demonstrate a highly specific function of the c-ros receptor tyrosine kinase during development of distinct epithelial cells.
Asunto(s)
Epidídimo/citología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Animales , Diferenciación Celular/fisiología , Línea Celular , Epidídimo/enzimología , Células Epiteliales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Espermatozoides/citologíaRESUMEN
Limb muscles develop from cells that migrate from the somites. The signal that induces migration of myogenic precursor cells to the limb emanates from the mesenchyme of the limb bud. Here we report that the c-met-encoded receptor tyrosine kinase is essential for migration of myogenic precursor cells into the limb anlage and for migration into diaphragm and tip of tongue. In c-met homozygous mutant (-/-) mouse embryos, the limb bud and diaphragm are not colonized by myogenic precursor cells and, as a consequence, skeletal muscles of the limb and diaphragm do not form. In contrast, development of the axial skeletal muscles proceeds in the absence of c-met signalling. The specific ligand of the c-met protein, the motility and growth factor scatter factor/hepatocyte growth factor, is expressed in limb mesenchyme and can thus provide the signal for migration which is received by c-met. We have therefore identified a paracrine signalling system that regulates migration of myogenic precursor cells.
Asunto(s)
Factor de Crecimiento de Hepatocito/fisiología , Músculos/embriología , Proteínas Tirosina Quinasas Receptoras/fisiología , Transducción de Señal , Animales , Movimiento Celular , Quimera , Diafragma/embriología , Extremidades/embriología , Mesodermo , Ratones , Músculo Esquelético/citología , Músculo Esquelético/embriología , Músculos/citología , Mutagénesis , Proteínas Proto-Oncogénicas c-met , Proteínas Tirosina Quinasas Receptoras/genética , Células Madre/citología , Lengua/embriologíaRESUMEN
Signals transduced by the met tyrosine kinase, which is the receptor for scatter factor/hepatocyte growth factor, are of major importance for the regulation of epithelial cell motility, morphogenesis, and proliferation. We report here that different sets of tyrosine residues in the cytoplasmic domain of the met receptor affect signal transduction in epithelial cells in a positive or negative fashion: mutation of the C-terminal tyrosine residues 13-16 (Y1311, Y1347, Y1354, and Y1363) reduced or abolished ligand-induced cell motility and branching morphogenesis. In contrast, mutation of the juxtamembrane tyrosine residue 2 (Y1001) produced constitutively mobile, fibroblastoid cells. Furthermore, the gain-of-function mutation of tyrosine residue 2 suppressed the loss-of-function mutations of tyrosine residue 15 or 16. The opposite roles of the juxtamembrane and C-terminal tyrosine residues may explain the suggested dual function of the met receptor in both epithelial-mesenchymal interactions and tumor progression.
Asunto(s)
Mutación Puntual , Proteínas Tirosina Quinasas Receptoras/metabolismo , Tirosina , Secuencia de Aminoácidos , Animales , Western Blotting , División Celular , Línea Celular , Membrana Celular/metabolismo , Movimiento Celular , Análisis Mutacional de ADN , Perros , Epitelio/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Riñón , Morfogénesis , Mutagénesis Sitio-Dirigida , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-met , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Factors that control epithelial growth, motility, and morphogenesis play important roles in malignancy and in normal development. Here we discuss the molecular nature and the function of two types of molecules that control the development and maintenance of epithelia: Components that regulate epithelial cell adhesion; and soluble factors and their receptors that regulate growth, motility, differentiation, and morphogenesis. In development, the establishment of epithelial cell characteristics and organization is crucially dependent on cell adhesion and the formation of functional adherens junctions. The integrity of adherens junctions is frequently disturbed late in tumor progression, and the resulting loss of epithelial characteristics correlates with the metastatic potential of carcinoma cells. Various soluble factors that induce epithelial growth, motility, or differentiation in cell culture, function via tyrosine kinase receptors. We concentrate here on receptors that are expressed exclusively or predominantly on epithelia, and on ligands that are derived from the mesenchyme. In development, these receptors and their ligands function in mesenchymal-epithelial interactions, which are known to govern growth, morphogenesis, and differentiation of epithelia. During tumor development, mutations or overexpression of the receptors are frequently observed; these alterations contribute to the development and progression of carcinomas.
Asunto(s)
Carcinoma/fisiopatología , Células Epiteliales , Sustancias de Crecimiento/fisiología , Animales , División Celular/fisiología , Movimiento Celular/fisiología , Humanos , Morfogénesis/fisiologíaRESUMEN
The Ca(2+)-dependent cell adhesion molecule E-cadherin functions in the establishment and maintenance of epithelial cell morphology during embryogenesis and adulthood. Downregulation or complete shut-down of E-cadherin expression and mutation of the gene are observed during the progression of tumors of epithelial origin (carcinomas) and correlate with the metastatic potential. We have introduced a targeted mutation into the E-cadherin gene by homologous recombination in mouse embryonic stem cells. The mutation removes E-cadherin sequences essential for Ca2+ binding and for adhesive function. These embryonic stem cells were used to generate mice carrying the mutation. Heterozygous mutant animals appear normal and are fertile. However, the homozygous mutation is not compatible with life: E-cadherin -/- embryos show severe abnormalities before implantation. Particularly, the adhesive cells of the morula dissociate shortly after compaction has occurred, and their morphological polarization is then destroyed. Interestingly, the blastomers are still able to form desmosomes and tight junctions at sites of distorted cell-cell contact. Thus, maternal E-cadherin suffices for initial compaction of the morula but not for further preimplantation development to occur.
Asunto(s)
Blastocisto/fisiología , Cadherinas/genética , Cadherinas/fisiología , Animales , Secuencia de Bases , Blastocisto/ultraestructura , Blastómeros/fisiología , Blastómeros/ultraestructura , Quimera , Desmosomas/ultraestructura , Desarrollo Embrionario y Fetal , Marcación de Gen , Homocigoto , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Mórula/citología , Mutación/fisiología , Células MadreRESUMEN
The ros gene was originally found because it can, when mutated, induce malignant transformation. The protooncogene encodes an orphan receptor tyrosine kinase. We report here the isolation and characterization of the mouse c-ros cDNA and, in addition, the biochemical characterization of the receptor. Both, the endogenous c-ros protein from embryonal tissues and the recombinant protein are glycosylated molecules with an apparent molecular weight of 260,000. Pulse-chase analysis in Sf9 cells demonstrates that the c-ros protein is synthesized as a single chain, uncleaved molecule. Since the specific ligand of c-ros is not known, a hybrid receptor (trk/c-ros) which transmits c-ros-specific signals in response to nerve growth factor (NGF) was used to study the biological activities. In NIH3T3 cells, this trk/c-ros hybrid induces growth, a fusiform cell shape, and loss of contact inhibition of growth. However, the active hybrid receptor cannot replace IL-3 as survival factor in 32D myeloid cells. Compared to other receptors, the active c-ros tyrosine kinase domain displays thus overlapping, but not identical signalling specificities.
Asunto(s)
Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Proteínas Tirosina Quinasas Receptoras/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Células Cultivadas , Ratones , Datos de Secuencia Molecular , Transducción de Señal , SpodopteraRESUMEN
Prolonged activation of protein kinase C (PKC) types alpha and beta by tumor-promoting phorbol esters leads to desensitization of the phorbol ester response, downregulation of protein kinase C activity and depletion of the protein kinase C polypeptide. When the gamma isoenzyme of PKC is transiently expressed in COS-1 cells and exposed to phorbol esters, PKC-Gamma is downregulated in COS cells although these cells do not normally express this subtype. A point mutation in the putative ATP-binding site (Lys-380----Met-380) of the protein kinase C gamma isoenzyme which results in a kinase-deficient enzyme does not interfere with this downregulation. Our results suggest that autophosphorylation or constitutive signalling through the protein kinase C-gamma kinase domain is not a prerequisite for downregulation of PKC activity.