RESUMEN
Replacement of indium tin oxide with the intrinsically conducting polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) has been of significant interest in recent years as a result of lower processing and material costs. In addition, the inclusion of additives has been reported to further enhance the conductivity, rheology, and wettability of PEDOT:PSS. In this study, Tween 80 was shown to decrease the sheet resistance of PEDOT:PSS films from approximately 1000 to 76 Ωâ¡-1 at a 2.67 wt% surfactant concentration. Through X-ray diffraction, Raman spectroscopy, and atomic force microscopy, it was shown that the surfactant caused phase separation and structural ordering of the PEDOT and PSS components, leading to this improvement in conductivity. Furthermore, Tween 80 altered the rheological properties and decreased the surface tension of PEDOT:PSS, making coating common commodity polymers, often used as flexible substrates, more viable.
RESUMEN
Developmentally regulated alternative splicing produces 'neonatal' and 'adult' isoforms of four Na(+) channels in human brain, NaV1.1, NaV1.2, NaV1.3 and NaV1.6. Heterologously expressed 'neonatal' NaV1.2 channels are less excitable than 'adult' channels; however, functional importance of this difference is unknown. We hypothesized that the 'neonatal' NaV1.2 may reduce neuronal excitability and have a seizure-protective role during early brain development. To test this hypothesis, we generated NaV1.2(adult) mice expressing only the 'adult' NaV1.2, and compared the firing properties of pyramidal cortical neurons, as well as seizure susceptibility, between the NaV1.2(adult) and wild-type (WT) mice at postnatal day 3 (P3), when the 'neonatal' isoform represents 65% of the WT NaV1.2. We show significant increases in action potential firing in NaV1.2(adult) neurons and in seizure susceptibility of NaV1.2(adult) mice, supporting our hypothesis. At postnatal day 15 (P15), when 17% of the WT NaV1.2 is 'neonatal', the firing properties of NaV1.2(adult) and WT neurons converged. However, inhibitory postsynaptic currents in NaV1.2(adult) neurons were larger and the expression level of Scn2a mRNA was 24% lower compared with the WT. The enhanced seizure susceptibility of the NaV1.2(adult) mice persisted into adult age. The adult NaV1.2(adult) mice also exhibited greater risk-taking behaviour. Overall, our data reveal a significant impact of 'neonatal' NaV1.2 on neuronal excitability, seizure susceptibility and behaviour and may contribute to our understanding of NaV1.2 roles in health and diseases such as epilepsy and autism.
Asunto(s)
Empalme Alternativo , Conducta Animal , Predisposición Genética a la Enfermedad/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Convulsiones/genética , Potenciales de Acción , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Exones , Masculino , Ratones , Ratones Endogámicos C57BL , Canal de Sodio Activado por Voltaje NAV1.2/genética , Neuronas/citología , Neuronas/metabolismo , Pentilenotetrazol/efectos adversos , Fenotipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Ubiquitin ligases of the Nedd4 family are important for axon and dendrite development, but little is known about their adaptor, Nedd4 family-interacting protein 1 (Ndfip1), that is responsible for their enzymatic activation. To study the function of Ndfip1 in cortical development, we generated a conditional knock-out (conditional KO) in neurons. The Ndfip1 conditional KO mice were viable; however, cortical neurons in the adult brain exhibited atrophic characteristics, including stunted dendritic arbors, blebbing of dendrites, and fewer dendritic spines. In electron micrographs, these neurons appeared shrunken with compacted somata and involutions of the nuclear membrane. In culture, Ndfip1 KO neurons exhibited exuberant sprouting suggesting loss of developmental control. Biochemical analysis of postsynaptic density (PSD) fractions from Ndfip1 KO cortical and hippocampal neurons showed that the postsynaptic proteins (Arc and PSD-95) were reduced compared with wild-type controls. In addition, the PI3 kinase/Akt signaling pathway was altered. These results indicate that Ndfip1, through its Nedd4 effectors, is important for the development of dendrites and dendritic spines in the cortex.
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Proteínas Portadoras/genética , Espinas Dendríticas/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de la Membrana/genética , Neocórtex , Células Piramidales/diagnóstico por imagen , Animales , Animales Recién Nacidos , Fraccionamiento Celular , Células Cultivadas , Homólogo 4 de la Proteína Discs Large , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Guanilato-Quinasas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neocórtex/citología , Neocórtex/embriología , Neocórtex/crecimiento & desarrollo , Nestina/genética , Nestina/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , UltrasonografíaRESUMEN
During development, protein substrates need to be removed and degraded when they are no longer required. The E3 ubiquitin ligases, including Nedd4 family proteins, are a major group of enzymes responsible for adding ubiquitin chains to protein substrates prior to their degradation. Ndfip1 (Nedd4 family-interacting protein 1) is an adaptor and activator for Nedd4-family ubiquitin ligases for increasing substrate specificity. To study Nedd4-mediated ubiquitination during cortical development, we have mapped the spatio-temporal dynamics of Ndfip1 protein expression by immunocytochemistry. Ndfip1 expression was observed from embryonic day 11 (E11.5) until adult stages. Its presence increased during the postnatal stages and peaked at postnatal day 7 (P7). Spatially, Ndfip1 was found in the ventricular and marginal zones during corticogenesis but also in the cortical plate and subplate during midstage cortical development (E15.5). Postnatally, Ndfip1 was expressed in all cortical neurons (but not in glial cells) and this expression was both ubiquitous and uniform across cortical layers involving both pyramidal and non-pyramidal neurons. This consistent but dynamic pattern of Ndfip1 expression in temporal and spatial domains of the cortical landscape is indicative of complex programs of protein ubiquitination during corticogenesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Corteza Cerebral/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Interneuronas/metabolismo , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Ubiquitina-Proteína Ligasas Nedd4 , Células Piramidales/metabolismo , UbiquitinaciónRESUMEN
PTEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regulator of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiological events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was translocated into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family-interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overexpression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1(-/-) fibroblasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival.
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Isquemia Encefálica/metabolismo , Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Neuronas/fisiología , Fosfohidrolasa PTEN/metabolismo , Animales , Isquemia Encefálica/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Supervivencia Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/fisiología , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ubiquitina-Proteína Ligasas Nedd4 , Fotoblanqueo , Transporte de Proteínas , Ubiquitina-Proteína Ligasas/fisiología , UbiquitinaciónRESUMEN
The Reelin signaling pathway is essential for proper cortical development, but it is unclear to whether Reelin function is primarily important for cortical layering or neuron migration. It has been proposed that Reelin is perhaps required only for somal translocation but not glial-dependent locomotion. This implies that the location of neurons responding to Reelin is restricted to the outer regions of the cortical plate (CP). To determine whether Reelin is required for migration outside of the CP, we used time-lapse imaging to track the behavior of cells undergoing locomotion in the germinal zones. We focused on the migratory activity in the ventricular/subventricular zones where the first transition of bipolar to multipolar migration occurs and where functional Reelin receptors are known to be expressed. Despite Reelin loss, neurons had no difficulty in undergoing radial migration and indeed displayed greater migratory speed. Additionally, compared with the wild-type, reeler neurons displayed altered trajectories with greater deviation from a radial path. These results suggest that Reelin loss has early consequences for migration in the germinal zones that are portrayed as defective radial trajectories and migratory speeds. Together, these abnormalities can give rise to the increased cell dispersion observed in the reeler cortex.
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Moléculas de Adhesión Celular Neuronal/deficiencia , Movimiento Celular/genética , Proteínas de la Matriz Extracelular/deficiencia , Neocórtex/citología , Proteínas del Tejido Nervioso/deficiencia , Neuronas/patología , Serina Endopeptidasas/deficiencia , Animales , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Ratones , Ratones Mutantes Neurológicos , Neocórtex/metabolismo , Neocórtex/patología , Proteínas del Tejido Nervioso/genética , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/metabolismo , Malformaciones del Sistema Nervioso/fisiopatología , Neuronas/citología , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Proteína Reelina , Serina Endopeptidasas/genética , Transmisión Sináptica/genéticaRESUMEN
Reelin is an important protein that is indispensable for cortical lamination. In the absence of Reelin, cortical layers fail to form due to inappropriate neuron migration and positioning. The inversion of cortical layers is attributed to failure of neurons to migrate past earlier-generated neurons although how Reelin-insufficiency causes this is unclear. The issue is complicated by recent studies showing that very little Reelin is required for cortical layering. To test how variation in the number of Reelin-producing cells is linked to cortical lamination, we have employed Reelin(+/+) <--> Reelin(-/-) chimeras in which the number of Reelin-expressing neurons is adjusted. We found that the Reeler phenotype was rescued in chimeras with a large contribution of Reelin(+/+) neurons; conversely in chimeras with a weak contribution by Reelin(+/+) neurons, the mutant phenotype remained. However, increasing the number of Reelin(+/+) neurons beyond an unknown threshold resulted in partial rescue, with the formation of a correctly layered secondary cortex lying on top of an inverted mutant cortex. Therefore, the development of cortical layers in the correct order requires a minimal level of Reelin protein to be present although paradoxically, this is insufficient to prevent the simultaneous formation of inverted cortical layers in the same hemisphere.
Asunto(s)
Tipificación del Cuerpo/genética , Moléculas de Adhesión Celular Neuronal/biosíntesis , Moléculas de Adhesión Celular Neuronal/deficiencia , Corteza Cerebral/anomalías , Corteza Cerebral/crecimiento & desarrollo , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/deficiencia , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/deficiencia , Neuronas/metabolismo , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/deficiencia , Quimera por Trasplante/genética , Animales , Animales Recién Nacidos , Moléculas de Adhesión Celular Neuronal/genética , Movimiento Celular/genética , Corteza Cerebral/metabolismo , Proteínas de la Matriz Extracelular/genética , Femenino , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/metabolismo , Neurogénesis/genética , Neuronas/patología , Proteína Reelina , Serina Endopeptidasas/genética , Quimera por Trasplante/crecimiento & desarrollo , Quimera por Trasplante/metabolismoRESUMEN
BACKGROUND: Seizure-related gene 6 (Sez-6) is expressed in neurons of the mouse brain, retina and spinal cord. In the cortex, Sez-6 plays a role in specifying dendritic branching patterns and excitatory synapse numbers during development. METHODOLOGY/PRINCIPAL FINDINGS: The distribution pattern of Sez-6 in the retina was studied using a polyclonal antibody that detects the multiple isoforms of Sez-6. Prominent immunostaining was detected in GABAergic, but not in AII glycinergic, amacrine cell subpopulations of the rat and mouse retina. Amacrine cell somata displayed a distinct staining pattern with the Sez-6 antibody: a discrete, often roughly triangular-shaped bright spot positioned between the nucleus and the apical dendrite superimposed over weaker general cytoplasmic staining. Displaced amacrines in the ganglion cell layer were also positive for Sez-6 and weaker staining was occasionally observed in neurons with the morphology of alpha ganglion cells. Two distinct Sez-6 positive strata were present in the inner plexiform layer in addition to generalized punctate staining. Certain inner nuclear layer cells, including bipolar cells, stained more weakly and diffusely than amacrine cells, although some bipolar cells exhibited a perinuclear "bright spot" similar to amacrine cells. In order to assess the role of Sez-6 in the retina, we analyzed the morphology of the Sez-6 knockout mouse retina with immunohistochemical markers and compared ganglion cell dendritic arbor patterning in Sez-6 null retinae with controls. The functional importance of Sez-6 was assessed by dark-adapted paired-flash electroretinography (ERG). CONCLUSIONS: In summary, we have reported the detailed expression pattern of a novel retinal marker with broad cell specificity, useful for retinal characterization in rodent experimental models. Retinal morphology, ganglion cell dendritic branching and ERG waveforms appeared normal in the Sez-6 knockout mouse suggesting that, in spite of widespread expression of Sez-6, retinal function in the absence of Sez-6 is not affected.
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Células Amacrinas/metabolismo , Eliminación de Gen , Proteínas del Tejido Nervioso/genética , Retina/metabolismo , Animales , Western Blotting , Electrorretinografía , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Fluorescente , Ratas , Ratas Sprague-Dawley , Retina/citología , Retina/fisiologíaRESUMEN
The factors affecting normal oligodendrocyte positioning in the cerebral cortex are unknown. Apart from the white matter, the highest numbers of oligodendrocytes in the rodent cortex are found in Layers V/VI, where the infragranular neurons normally reside. Few, if any, oligodendrocytes are normally found in the superficial cortical layers. To test whether or not this asymmetric positioning of oligodendrocytes is linked to the lamina positions of Layer V/VI projection neurons, mutant mice that cause neuronal layer inversion were examined. In three lines of mutant mice (Reeler, disabled-1, and p35) examined, representing two different genetic signaling pathways, the oligodendrocyte distribution was altered from an asymmetric to a symmetric distribution pattern. Unlike cortical neurons that are inverted in these mutant mice, the lack of oligodendrocyte inversion suggests a decoupling of the genetic mechanisms governing neuronal versus oligodendrocyte patterning. We conclude that oligodendrocyte positioning is not linked to the layer positions of V/VI projection neurons.
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Corteza Cerebral/anatomía & histología , Corteza Cerebral/citología , Neuronas/citología , Oligodendroglía/citología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Moléculas de Adhesión Celular Neuronal/genética , Corteza Cerebral/metabolismo , Proteínas de la Matriz Extracelular/genética , Inmunohistoquímica , Ratones , Ratones Mutantes , Ratones Mutantes Neurológicos , Fibras Nerviosas Mielínicas/fisiología , Proteínas del Tejido Nervioso/genética , Neuronas/fisiología , Oligodendroglía/metabolismo , Fosfotransferasas/genética , Proteína Reelina , Serina Endopeptidasas/genéticaRESUMEN
Development of appropriate dendritic arbors is crucial for neuronal information transfer. We show, using seizure-related gene 6 (sez-6) null mutant mice, that Sez-6 is required for normal dendritic arborization of cortical neurons. Deep-layer pyramidal neurons in the somatosensory cortex of sez-6 null mice exhibit an excess of short dendrites, and cultured cortical neurons lacking Sez-6 display excessive neurite branching. Overexpression of individual Sez-6 isoforms in knockout neurons reveals opposing actions of membrane-bound and secreted Sez-6 proteins, with membrane-bound Sez-6 exerting an antibranching effect under both basal and depolarizing conditions. Layer V pyramidal neurons in knockout brain slices show reduced excitatory postsynaptic responses and a reduced dendritic spine density, reflected by diminished punctate staining for postsynaptic density 95 (PSD-95). In behavioral tests, the sez-6 null mice display specific exploratory, motor, and cognitive deficits. In conclusion, cell-surface protein complexes involving Sez-6 help to sculpt the dendritic arbor, in turn enhancing synaptic connectivity.
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Corteza Cerebral/anomalías , Corteza Cerebral/citología , Dendritas/ultraestructura , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas del Tejido Nervioso/genética , Células Piramidales/citología , Animales , Diferenciación Celular/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Corteza Cerebral/metabolismo , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/fisiopatología , Dendritas/metabolismo , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Homólogo 4 de la Proteína Discs Large , Potenciales Postsinápticos Excitadores/genética , Femenino , Guanilato-Quinasas , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/metabolismo , Malformaciones del Sistema Nervioso/fisiopatología , Vías Nerviosas/anomalías , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Células Piramidales/metabolismo , Transmisión Sináptica/genéticaRESUMEN
How is the activation of Reelin signalling within neurons translated into the layering of cortical neurons? To address this question, we made mouse chimeras to test the reciprocal effects of neurons possessing different genotypes but sharing a common cortical environment during development. In chimeras composed of wild-type and mutant neurons (for either Reelin, Dab1 or p35 genes), a common observation was the formation of a second set of cortical layers on top of an inverted mutant cortex. The secondary cortex was invariably layered in the correct order, and in Dab1 and p35 chimeras, they were principally composed of wild-type neurons. In contrast to these cell-autonomous effects, Reelin chimeras displayed non cell-autonomous effects. In these chimeras, only a small number of wild-type neurons were required to be present in order for a secondary cortex to be formed. Interestingly, the principal constituents of the secondary cortex are not wild-type but mutant neurons, suggesting non cell-autonomous signalling by low levels of Reelin. Overall, these results suggest that information for the generation of cortical layers is vested within neuroepithelial progenitors even before the first neurons have been born, but the guidance of successive generations of daughter neurons to their proper locations requires the activation of Reelin and p35.
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Corteza Cerebral/embriología , Quimera/embriología , Neuronas/fisiología , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/fisiología , Movimiento Celular/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/fisiología , Ratones , Ratones Mutantes Neurológicos , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Proteína Reelina , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/fisiología , Transducción de SeñalRESUMEN
We tested the response of interneurons to the absence of Reelin signaling or p35 in the mouse neocortex. We provide three independent strands of evidence to demonstrate that layering of late-born (but not early-born) interneurons is regulated by Reelin signaling. First, early-born and late-born interneurons behaved differently in mice lacking Reelin or disabled 1 (Dab1). Early-born interneurons showed layer inversion, whereas late-born interneurons did not demonstrate layer inversion but were randomly distributed across the cortex. Second, in p35 mutant brains (in which Reelin signaling is intact), late-born interneurons are appropriately positioned in the upper layers despite the malpositioning of all other cortical neurons in these mice. Third, transplanted late-born interneuron precursors (wild type) into Dab1(-/-) cortices showed appropriate upper layer segregation. Together, these results indicate that, in the absence of Reelin signaling, late-born interneurons fail to laminate properly, and this is restored in an environment in which Reelin signaling is intact. These studies suggest different mechanisms for the stratification of cortical interneurons. Whereas the early-born interneurons appear to be associated with projection neuron layering, late-born interneurons rely on Reelin signaling for their correct lamination.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/fisiología , Proteínas de la Matriz Extracelular/fisiología , Interneuronas/citología , Neocórtex/citología , Neocórtex/embriología , Proteínas del Tejido Nervioso/fisiología , Fosfotransferasas/fisiología , Serina Endopeptidasas/fisiología , Animales , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Interneuronas/enzimología , Interneuronas/trasplante , Masculino , Ratones , Ratones Noqueados , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/genética , Fosfotransferasas/genética , Proteína Reelina , Serina Endopeptidasas/genética , Transducción de SeñalRESUMEN
UNLABELLED: The PTHrP gene generates low-abundance mRNA and protein products that are not easily localized by in situ hybridization histochemistry or immunohistochemistry. We report here a PTHrP-lacZ knockin mouse in which beta-gal activity seems to provide a simple and sensitive read-out of PTHrP gene expression. INTRODUCTION: PTH-related protein (PTHrP) is widely expressed in fetal and adult tissues, typically as low-abundance mRNA and protein products that maybe difficult to localize by conventional methods. We created a PTHrP-lacZ knockin mouse as a means of surveying PTHrP gene expression in general and of identifying previously unrecognized sites of PTHrP expression. MATERIALS AND METHODS: We created a lacZ reporter construct under the control of endogenous PTHrP gene regulatory sequences. The AU-rich instability sequences in the PTHrP 3' untranslated region (UTR) were replaced with SV40 sequences, generating products with lacZ/beta gal kinetics rather than those of PTHrP. A nuclear localization sequence was not present in the construct. RESULTS: We characterized beta-galactosidase (beta-gal) activity in embryonic whole mounts and in the skeleton in young and adult animals. In embryos, we confirmed widespread PTHrP expression in many known sites and in several novel epidermal appendages (nail beds and footpads). In costal cartilage, beta-gal activity localized to the perichondrium but not the underlying chondrocytes. In the cartilaginous molds of forming long bones, beta-gal activity was first evident at the proximal and distal ends. Shortly after birth, the developing secondary ossification center formed in the center of this PTHrP-rich chondrocyte population. As the secondary ossification center developed, it segregated this population into two distinct PTHrP beta-gal+ subpopulations: a subarticular subpopulation immediately subjacent to articular chondrocytes and a proliferative chondrocyte subpopulation proximal to the chondrocyte columns in the growth plate. These discrete populations remained into adulthood. beta-gal activity was not identified in osteoblasts but was present in many periosteal sites. These included simple periosteum as well as fibrous tendon insertion sites of the so-called bony and periosteal types; the beta-gal-expressing cells in these sites were in the outer fibrous layer of the periosteum or its apparent equivalents at tendon insertion sites. Homozygous PTHrP-lacZ knockin mice had the expected chondrodysplastic phenotype and a much expanded region of proximal beta-gal activity in long bones, which appeared to reflect in large part the effects of feedback signaling by Indian hedgehog on proximal cell proliferation and PTHrP gene expression. CONCLUSIONS: The PTHrP-lacZ mouse seems to provide a sensitive reporter system that may prove useful as a means of studying PTHrP gene expression.
Asunto(s)
Desarrollo Óseo/fisiología , Regulación del Desarrollo de la Expresión Génica , Operón Lac , Proteína Relacionada con la Hormona Paratiroidea/biosíntesis , Animales , Huesos/citología , Huesos/embriología , Proliferación Celular , Condrocitos/citología , Condrocitos/metabolismo , Marcadores Genéticos/genética , Ratones , Ratones Transgénicos , Especificidad de Órganos , Osteoblastos/citología , Osteoblastos/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/genética , Transgenes/genéticaRESUMEN
Bone development is regulated by conserved signalling pathways that are linked to multifunctional growth factors and their high affinity receptors. Parathyroid hormone-related peptide (PTHrP) and fibroblast growth factor receptor 3 (FGFR3) have been shown to play pivotal, and sometimes complementary, roles in the replication, maturation and death of chondrocytes during endochondral bone formation. To gain further insight into how these pathways coordinate cartilage and bone development, we generated mice lacking expression of both PTHrP and FGFR3. The phenotype of compound mutant mice resembled that of their PTHrP-deficient littermates with respect to neonatal lethality, facial dysmorphism and foreshortening of the limbs. The absence of PTHrP in the developing epiphyseal cartilage of PTHrP-/- and PTHrP-/-/FGFR3-/- mice resulted in a dominant hypo-proliferative phenotype. However, abnormalities such as the presence of nonhypertrophic cells among hypertrophic chondrocytes and excessive apoptosis seen in the hypertrophic zone of PTHrP-/- mice were absent in the PTHrP-/-/FGFR3-/- mice. Furthermore, the absence of FGFR3 in single and compound mutant mice led to decreased expression of vascular endothelial growth factor (VEGF) and an increase in depth of hypertrophic chondrocytes. These observations indicate that FGFR3 deficiency can rescue some of the defects seen in PTHrP-deficient mice and that it plays an important role in the regulation of chondrocyte differentiation and hypertrophy. These studies support a dominant role for PTHrP in regulating the pool of proliferating cells during limb development and suggest that signalling by FGFR3 plays a more prominent role in cartilage maturation and vascular invasion at the chondro-osseous junction.
Asunto(s)
Desarrollo Óseo , Cartílago/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Animales , Animales Recién Nacidos , Apoptosis , Cartílago/patología , Colágeno Tipo II/metabolismo , Colágeno Tipo X/metabolismo , Placa de Crecimiento/metabolismo , Placa de Crecimiento/patología , Hiperostosis/genética , Hiperostosis/metabolismo , Hiperostosis/patología , Inmunohistoquímica , Ratones , Mutación/genética , Proteína Relacionada con la Hormona Paratiroidea/genética , Fenotipo , Proteínas Tirosina Quinasas/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Tibia/metabolismo , Tibia/patología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The migration, arrest, and ultimately positioning of cortical neurons require signaling activity from Reelin as well as from cyclin-dependent kinase 5 (Cdk5). Although both molecules control neuronal positioning, they achieve their effects by quite separate molecular pathways. Cdk5 is a serine-threonine kinase, the activity of which is dependent on its activating subunits p35 and p39. Mice deficient in Cdk5, p35, or both p35 and p39 display the hallmarks of disturbed cortical development, including cortical layer inversion, neuronal disorientation, and abnormal fiber infiltration. To distinguish between the cell- and non cell-autonomous functions of p35, we constructed p35+/+ <--> p35-/- chimeras using the lacZ gene as an independent marker for p35+/+ cells. In this shared developmental space, wild-type and mutant neurons behaved cell-autonomously with respect to layering. Wild-type cells formed a properly layered supercortex that is mirrored by an inverted mutant cortex lying underneath. However, this genotype-specific behavior was confined to the pyramidal population, and interneurons belonging to either genotype were indiscriminately distributed. However, there was also non cell-autonomous rescue of mutant neurons, and this rescue was specific only to early-born pyramidal neurons belonging to layer V. Rescued neurons reached the correct layer address and possessed appropriate neuronal morphology, orientation, and projections. Later-born neurons belonging to layers II and III were not rescued. These results demonstrate that p35 signaling can have both cell- and non cell-autonomous consequences, and their effects are not uniformly shared by cortical neurons born at different times or born at different places (projection neurons vs interneurons).
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Corteza Cerebral/citología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Animales , Movimiento Celular , Corteza Cerebral/crecimiento & desarrollo , Quimera , Interneuronas/citología , Ratones , Ratones Noqueados , Fibras Nerviosas/ultraestructura , Proteínas del Tejido Nervioso/genética , Células Piramidales/citología , Proteína ReelinaRESUMEN
Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.
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Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Animales , Animales Recién Nacidos , Formación de Anticuerpos , Femenino , Sistema Inmunológico/crecimiento & desarrollo , Técnicas In Vitro , Activación de Linfocitos , Activación de Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Células Progenitoras Mieloides/citología , Proteínas Nucleares/deficiencia , Osteoclastos/citología , Fenotipo , Transducción de Señal , Factor de Necrosis Tumoral alfa/fisiología , Ubiquitina-Proteína LigasasRESUMEN
Parathyroid hormone-related protein (PTHrP) null mutant mice were analyzed to investigate an additional role for PTHrP in cell differentiation. We found ectopic cartilage formation in the mandibular coronoid process in newborn mice. While many previous studies involving PTHrP gene knockout mouse have shown that the cartilage in various regions becomes smaller, this is the first report showing an "increase" of cartilage volume. Investigations of mandibular growth using normal mice indicated that coronoid secondary cartilage never formed from E 15 to d 4, but small amount of cartilage temporally formed at d 7, and this also applies to PTHrP-wild type mice. Therefore, PTHrP deficiency consequently advanced the secondary cartilage formation, which is a novel role of PTHrP in chondrocyte differentiation. In situ hybridization of matrix proteins showed that this coronoid cartilage had characteristics of the lower hypertrophic cell zone usually present at the site of endochondral bone formation and/or "chondroid bone" occasionally found in distraction osteogenesis. In addition, the coronoid process in the PTHrP-deficient mouse also showed abnormal expansion of bone marrow and an increase in the number of multinucleated osteoclasts, an indication of abnormal bone modeling. These results indicate that PTHrP is involved in bone modeling as well as in chondrocyte differentiation. In situ hybridization of matrix protein mRNAs in the abnormal mandibular condylar cartilage revealed that this cartilage was proportionally smaller, supporting previous immunohistochemical results.
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Cartílago/anomalías , Cartílago/patología , Coristoma/patología , Mandíbula/anomalías , Mandíbula/patología , Proteína Relacionada con la Hormona Paratiroidea/deficiencia , Animales , Animales Recién Nacidos , Médula Ósea/patología , Matriz Ósea/metabolismo , Remodelación Ósea/genética , Cartílago/metabolismo , Condrocitos/metabolismo , Condrocitos/patología , Coristoma/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Inmunohistoquímica , Masculino , Mandíbula/metabolismo , Ratones , Ratones Noqueados , Osteoclastos/patología , Osteogénesis/genética , Proteína Relacionada con la Hormona Paratiroidea/genética , ARN Mensajero/metabolismoRESUMEN
In a previous study, it was shown that tooth germs of neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are penetrated or compressed by the surrounding alveolar bone, suggesting an important role for PTHrP in the formation and activation of osteoclasts around growing tooth germs. In order to elucidate the role of PTHrP during the development of the tooth germ and related structures, mandibular explants containing cap stage tooth germs of embryonic day 14, homozygous mice were here cultured with or without surrounding alveolar bone. There was no difference in the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells around the first molars of homozygous and wild-type mice. After 10 days of culture, osteoclastic cells were rarely present in explants from homozygous mice and penetration of alveolar bone into the dental papilla was observed. The decline in osteoclast number was partly restored by the addition of PTHrP to the culture. Tooth germs of both wild-type and homozygous mice cultured without alveolar bone developed well, with no apparent structural abnormality; dentine formation was evident after 10 days. These data suggest that PTHrP is not required for the development of the tooth germ proper but is indispensable in promoting the osteoclast formation required to accommodate that development.