RESUMEN
Congenital stationary night blindness (CSNB) is an inherited retinal disease that causes a profound loss of rod sensitivity without severe retinal degeneration. One well-studied rhodopsin point mutant, G90D-Rho, is thought to cause CSNB because of its constitutive activity in darkness causing rod desensitization. However, the nature of this constitutive activity and its precise molecular source have not been resolved for almost 30 y. In this study, we made a knock-in (KI) mouse line with a very low expression of G90D-Rho (equal in amount to ~0.1% of normal rhodopsin, WT-Rho, in WT rods), with the remaining WT-Rho replaced by REY-Rho, a mutant with a very low efficiency of activating transducin due to a charge reversal of the highly conserved ERY motif to REY. We observed two kinds of constitutive noise: one being spontaneous isomerization (R*) of G90D-Rho at a molecular rate (R* s-1) 175-fold higher than WT-Rho and the other being G90D-Rho-generated dark continuous noise comprising low-amplitude unitary events occurring at a very high molecular rate equivalent in effect to ~40,000-fold of R* s-1 from WT-Rho. Neither noise type originated from G90D-Opsin because exogenous 11-cis-retinal had no effect. Extrapolating the above observations at low (0.1%) expression of G90D-Rho to normal disease exhibited by a KI mouse model with RhoG90D/WTand RhoG90D/G90D genotypes predicts the disease condition very well quantitatively. Overall, the continuous noise from G90D-Rho therefore predominates, constituting the major equivalent background light causing rod desensitization in CSNB.
Asunto(s)
Enfermedades Hereditarias del Ojo , Enfermedades Genéticas Ligadas al Cromosoma X , Miopía , Ceguera Nocturna , Rodopsina , Animales , Ceguera Nocturna/genética , Ceguera Nocturna/metabolismo , Enfermedades Hereditarias del Ojo/genética , Enfermedades Hereditarias del Ojo/metabolismo , Ratones , Rodopsina/genética , Rodopsina/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Miopía/genética , Miopía/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Oscuridad , Transducina/genética , Transducina/metabolismo , Técnicas de Sustitución del Gen , Modelos Animales de EnfermedadRESUMEN
Although olfactory mucosa possesses long-lived horizontal basal stem cells (HBCs) and remarkable regenerative capacity, the function of human olfactory neuroepithelium is significantly impaired in chronic inflammatory rhinosinusitis. Here, we show that, while inflammation initially damages olfactory neurons and activates HBC-mediated regeneration, continued inflammation locks HBCs in an undifferentiated state. Global gene expression in mouse HBCs reveals broad upregulation of NF-κB-regulated cytokines and chemokines including CCL19, CCL20, and CXCL10, accompanied by enhancement of "stemness"-related transcription factors. Loss-of-function studies identify an NF-κB-dependent role of HBCs in amplifying inflammatory signaling, contributing to macrophage and T cell local proliferation. Chronically activated HBCs signal macrophages to maintain immune defense and prevent Treg development. In diseased human olfactory tissue, activated HBCs in a P63+ undifferentiated state similarly contribute to inflammation through chemokine production. These observations establish a mechanism of chronic rhinosinusitis-associated olfactory loss, caused by a functional switch of neuroepithelial stem cells from regeneration to immune defense.
Asunto(s)
Inflamación/inmunología , Neuronas/fisiología , Mucosa Olfatoria/fisiología , Rinitis/inmunología , Sinusitis/inmunología , Células Madre/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Autorrenovación de las Células/genética , Células Cultivadas , Enfermedad Crónica , Humanos , Inmunidad , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , RegeneraciónRESUMEN
The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double stranded) engage in a high-efficiency HDR mechanism that requires only â¼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1,000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand annealing. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.
Asunto(s)
Proteínas Bacterianas/metabolismo , Reparación del ADN , Endonucleasas/metabolismo , Edición Génica/métodos , Animales , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Roturas del ADN de Doble Cadena , Células HEK293 , Humanos , Ratones , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido NucleicoRESUMEN
Adult neural stem cells/progenitor cells residing in the basal layer of the olfactory epithelium are capable of reconstituting the neuroepithelium even after severe damage. The molecular events underlying this regenerative capacity remain elusive. Here we show that the repair of neuroepithelium after lesioning is accompanied by an acute, but self-limited, inflammatory process. Attenuation of inflammatory cell recruitment and cytokine production by dexamethasone impairs proliferation of progenitor horizontal basal cells (HBCs) and subsequent neuronal differentiation. Using TNF-α receptor-deficient mice, we identify TNF-α signaling as an important contributor to this inflammatory and reparative process, mainly through TNF-α receptor 1. HBC-selective genetic ablation of RelA (p65), the transcriptional activator of the NF-κB pathway, retards inflammation and impedes proliferation at the early stages of regeneration and suggests HBCs directly participate in cross-talk between immune response and neurogenesis. Loss of RelA in the regenerating neuroepithelium perturbs the homeostasis between proliferation and apoptosis while enhancing JNK signaling. Together, our results support a model in which acute inflammation after injury initiates important regenerative signals in part through NF-κB-mediated signaling that activates neural stem cells to reconstitute the olfactory epithelium.
Asunto(s)
Regeneración Nerviosa , Mucosa Olfatoria/inmunología , Factor de Transcripción ReIA/metabolismo , Animales , Inflamación/metabolismo , Ratones Noqueados , Mucosa Olfatoria/metabolismo , Factor de Transcripción ReIA/genéticaRESUMEN
The molecular circadian clocks in the mammalian retina are locally synchronized by environmental light cycles independent of the suprachiasmatic nuclei (SCN) in the brain. Unexpectedly, this entrainment does not require rods, cones, or melanopsin (OPN4), possibly suggesting the involvement of another retinal photopigment. Here, we show that the ex vivo mouse retinal rhythm is most sensitive to short-wavelength light but that this photoentrainment requires neither the short-wavelength-sensitive cone pigment [S-pigment or cone opsin (OPN1SW)] nor encephalopsin (OPN3). However, retinas lacking neuropsin (OPN5) fail to photoentrain, even though other visual functions appear largely normal. Initial evidence suggests that OPN5 is expressed in select retinal ganglion cells. Remarkably, the mouse corneal circadian rhythm is also photoentrainable ex vivo, and this photoentrainment likewise requires OPN5. Our findings reveal a light-sensing function for mammalian OPN5, until now an orphan opsin.
Asunto(s)
Córnea/fisiología , Proteínas de la Membrana/fisiología , Opsinas/fisiología , Retina/fisiología , Núcleo Supraquiasmático/fisiología , Animales , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Opsinas/genética , Rayos UltravioletaRESUMEN
In a classic model of mammalian brain formation, precursors of principal glutamatergic neurons migrate radially along radial glia fibers whereas GABAergic interneuron precursors migrate tangentially. These migration modes have significant implications for brain function. Here we used clonal lineage tracing of active radial glia-like neural stem cells in the adult mouse dentate gyrus and made the surprising discovery that proliferating neuronal precursors of glutamatergic granule neurons exhibit significant tangential migration along blood vessels, followed by limited radial migration. Genetic birthdating and morphological and molecular analyses pinpointed the neuroblast stage as the main developmental window when tangential migration occurs. We also developed a partial "whole-mount" dentate gyrus preparation and observed a dense plexus of capillaries, with which only neuroblasts, among the entire population of progenitors, are directly associated. Together, these results provide insight into neuronal migration in the adult mammalian nervous system.
Asunto(s)
Encéfalo/metabolismo , Giro Dentado/fisiología , Glutamina/química , Neurogénesis/fisiología , Neuronas/fisiología , Animales , Mapeo Encefálico/métodos , Movimiento Celular , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/fisiología , Imagenología Tridimensional , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Microscopía Fluorescente , Modelos Neurológicos , Células-Madre Neurales/citología , Neuronas/citologíaRESUMEN
Persistent neurogenesis in the olfactory epithelium provides a unique model to study neural stem cell self-renewal and fate determination. In the olfactory neuroepithelium, globose basal cells (GBCs) are considered to be the direct progenitors of olfactory neurons. However, the study of neurogenesis from GBCs has been impeded by the paucity of GBC-specific markers. Here we report that Lgr5, a recently discovered adult stem cell marker, is exclusively expressed in GBCs in neonatal and adult mice. Lgr5(+) cells display characteristics of cycling stem cells, including Ki67 expression and EdU incorporation. Lineage tracing analysis demonstrates that Lgr5(+) GBCs regenerate multiple cell types under normal turnover condition or after olfactory lesion. Furthermore, upregulation or downregulation of Wnt signaling in vivo indicates a key role of Wnt signaling not only in maintaining Lgr5(+) cell proliferation and promoting neuroregeneration, but also in delaying sensory neuron maturation. Together, our observations provided new insights into the dynamics of neurogenesis in the olfactory epithelium.
Asunto(s)
Células Madre Multipotentes/fisiología , Neuronas Receptoras Olfatorias/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Animales Recién Nacidos , Cápsulas Bacterianas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Citometría de Flujo , Proteína GAP-43/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno Ki-67/metabolismo , Ratones , Ratones Transgénicos , Proteína Marcadora Olfativa/metabolismo , Mucosa Olfatoria/citología , Receptores Acoplados a Proteínas G/genética , beta-Galactosidasa/metabolismoRESUMEN
The subventricular zone (SVZ) continuously supplies new interneurons that incorporate into pre-existing olfactory bulb circuitry. Khodosevich et al. (2013) show that connective tissue growth factor (CTGF) regulates a multicellular signaling cascade determining the number of postnatally born inhibitory interneurons in odor-activated glomeruli.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Regulación de la Expresión Génica/genética , Interneuronas/fisiología , Bulbo Olfatorio/citología , Olfato/genética , Animales , Femenino , Humanos , MasculinoRESUMEN
The basic scheme of odor perception and signaling from olfactory cilia to the brain is well understood. However, factors that affect olfactory acuity of an animal, the threshold sensitivity to odorants, are less well studied. Using signal sequence trap screening of a mouse olfactory epithelium cDNA library, we identified a novel molecule, Goofy, that is essential for olfactory acuity in mice. Goofy encodes an integral membrane protein with specific expression in the olfactory and vomeronasal sensory neurons and predominant localization to the Golgi compartment. Goofy-deficient mice display aberrant olfactory phenotypes, including the impaired trafficking of adenylyl cyclase III, stunted olfactory cilia, and a higher threshold for physiological and behavioral responses to odorants. In addition, the expression of dominant-negative form of cAMP-dependent protein kinase results in shortening of olfactory cilia, implying a possible mechanistic link between cAMP and ciliogenesis in the olfactory sensory neurons. These results demonstrate that Goofy plays an important role in establishing the acuity of olfactory sensory signaling.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Odorantes , Vías Olfatorias/metabolismo , Neuronas Receptoras Olfatorias/fisiología , Transducción de Señal/fisiología , Adenilil Ciclasas/metabolismo , Animales , Clonación Molecular , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Potenciales Evocados/genética , Proteínas de Unión al GTP/deficiencia , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica/genética , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Marcadora Olfativa/genética , Proteína Marcadora Olfativa/metabolismo , Vías Olfatorias/anatomía & histología , ARN Mensajero , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Análisis de Secuencia , Transducción de Señal/genética , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The master transcription factor Pparγ regulates the general differentiation program of both brown and white adipocytes. However, it has been unclear whether Pparγ also controls fat lineage-specific characteristics. Here, we show that early B cell factor-2 (Ebf2) regulates Pparγ binding activity to determine brown versus white adipocyte identity. The Ebf DNA-binding motif was highly enriched within brown adipose-specific Pparγ binding sites that we identified by genome-wide ChIP-Seq. Of the Ebf isoforms, Ebf2 was selectively expressed in brown relative to white adipocytes and was bound at brown adipose-specific Pparγ target genes. When expressed in myoblasts or white preadipose cells, Ebf2 recruited Pparγ to its brown-selective binding sites and reprogrammed cells to a brown fat fate. Brown adipose cells and tissue from Ebf2-deficient mice displayed a loss of brown-specific characteristics and thermogenic capacity. Together, these results identify Ebf2 as a key transcriptional regulator of brown fat cell fate and function.
Asunto(s)
Adipocitos Marrones/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Adipocitos Marrones/citología , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sitios de Unión , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Ratones Noqueados , PPAR gamma/metabolismo , Unión Proteica , Isoformas de Proteínas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Zfp423/OAZ, a multi-zinc finger protein, is proposed to participate in neuronal differentiation through interactions with the Olf/EBF (O/E) family of transcription factors and mediate extrinsic BMP signaling pathways. These activities are associated with distinct domains of the Olf/EBF-associated zinc finger (OAZ) protein. Sustained OAZ expression arrests olfactory sensory neurons (OSNs) at an immature state and alters olfactory receptor expression, but the mechanism remains elusive. We show here that constitutive expression of a C-terminal mutant OAZ (OAZΔC) in mice that selectively disrupts OAZ-O/E interaction while retaining other activities, exhibits apparently normal OSN differentiation. Additionally, interfering with potential BMP signaling pathways by inducible Follistatin expression in adult mice does not alter the neuronal lineage or differentiation status. Our results indicate that O/E-mediated processes are essential for the differentiation of OSNs and the establishment of a mature phenotype. BMP signaling pathways, if they are active in normal adult olfactory epithelium, may play a minor role in this tissue.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteínas de Unión al ADN/genética , Neurogénesis/genética , Neuronas Receptoras Olfatorias/citología , Mutación Puntual , Receptores Odorantes/fisiología , Factores de Transcripción/genética , Transcripción Genética , Dedos de Zinc/genética , Animales , Proteínas Morfogenéticas Óseas/fisiología , Linaje de la Célula , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Folistatina/biosíntesis , Folistatina/genética , Folistatina/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Secuencias Hélice-Asa-Hélice , Ratones , Ratones Endogámicos C57BL , Mucosa Olfatoria/citología , Neuronas Receptoras Olfatorias/metabolismo , Fenotipo , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Receptores Odorantes/genética , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal/fisiología , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/fisiología , Dedos de Zinc/fisiologíaRESUMEN
Cilia are evolutionarily conserved microtubule-based organelles that are crucial for diverse biological functions, including motility, cell signaling and sensory perception. In humans, alterations in the formation and function of cilia manifest clinically as ciliopathies, a growing class of pleiotropic genetic disorders. Despite the substantial progress that has been made in identifying genes that cause ciliopathies, therapies for these disorders are not yet available to patients. Although mice with a hypomorphic mutation in the intraflagellar transport protein IFT88 (Ift88Tg737Rpw mice, also known as ORPK mice)5 have been well studied, the relevance of IFT88 mutations to human pathology is unknown. We show that a mutation in IFT88 causes a hitherto unknown human ciliopathy. In vivo complementation assays in zebrafish and mIMCD3 cells show the pathogenicity of this newly discovered allele. We further show that ORPK mice are functionally anosmic as a result of the loss of cilia on their olfactory sensory neurons (OSNs). Notably, adenoviral-mediated expression of IFT88 in mature, fully differentiated OSNs of ORPK mice is sufficient to restore ciliary structures and rescue olfactory function. These studies are the first to use in vivo therapeutic treatment to reestablish cilia in a mammalian ciliopathy. More broadly, our studies indicate that gene therapy is a viable option for cellular and functional rescue of the complex ciliary organelle in established differentiated cells.
Asunto(s)
Cilios/genética , Cilios/patología , Enfermedades Genéticas Congénitas/genética , Terapia Genética/métodos , Neuronas Receptoras Olfatorias/citología , Olfato/fisiología , Proteínas Supresoras de Tumor/genética , Adenoviridae , Animales , Prueba de Complementación Genética , Enfermedades Genéticas Congénitas/patología , Enfermedades Genéticas Congénitas/terapia , Vectores Genéticos , Humanos , Ratones , Microscopía Fluorescente , Mutación/genética , Neuronas Receptoras Olfatorias/metabolismo , Olfato/genética , Tubulina (Proteína)/metabolismo , Pez CebraRESUMEN
Nearly every ciliated organism possesses three B9 domain-containing proteins: MKS1, B9D1, and B9D2. Mutations in human MKS1 cause Meckel syndrome (MKS), a severe ciliopathy characterized by occipital encephalocele, liver ductal plate malformations, polydactyly, and kidney cysts. Mouse mutations in either Mks1 or B9d2 compromise ciliogenesis and result in phenotypes similar to those of MKS. Given the importance of these two B9 proteins to ciliogenesis, we examined the role of the third B9 protein, B9d1. Mice lacking B9d1 displayed polydactyly, kidney cysts, ductal plate malformations, and abnormal patterning of the neural tube, concomitant with compromised ciliogenesis, ciliary protein localization, and Hedgehog (Hh) signal transduction. These data prompted us to screen MKS patients for mutations in B9D1 and B9D2. We identified a homozygous c.301A>C (p.Ser101Arg) B9D2 mutation that segregates with MKS, affects an evolutionarily conserved residue, and is absent from controls. Unlike wild-type B9D2 mRNA, the p.Ser101Arg mutation failed to rescue zebrafish phenotypes induced by the suppression of b9d2. With coimmunoprecipitation and mass spectrometric analyses, we found that Mks1, B9d1, and B9d2 interact physically, but that the p.Ser101Arg mutation abrogates the ability of B9d2 to interact with Mks1, further suggesting that the mutation compromises B9d2 function. Our data indicate that B9d1 is required for normal Hh signaling, ciliogenesis, and ciliary protein localization and that B9d1 and B9d2 are essential components of a B9 protein complex, disruption of which causes MKS.
Asunto(s)
Trastornos de la Motilidad Ciliar/genética , Encefalocele/genética , Enfermedades Renales Poliquísticas/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Análisis Mutacional de ADN , Ligamiento Genético , Homocigoto , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Tubo Neural/anomalías , Fenotipo , Polidactilia/genética , Transporte de Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retinitis Pigmentosa , Transducción de Señal , Pez Cebra/genéticaRESUMEN
Bardet-Biedl syndrome (BBS) is a pleiotropic, heterogeneous human disease whose etiology lies primarily in dysfunctional basal bodies and/or cilia. Both BBS patients and several BBS mouse models exhibit impaired olfactory function. To explore the nature of olfactory defects in BBS, a genetic ablation of the mouse Bbs8 gene that incorporates a fluorescent reporter protein was created. The endogenous BBS8 protein and reporter are particularly abundant in olfactory sensory neurons (OSNs), and specific BBS8 antibodies reveal staining in the dendritic knob in a shell-like structure that surrounds the basal bodies. Bbs8-null mice have reduced olfactory responses to a number of odorants, and immunohistochemical analyses reveal a near-complete loss of cilia from OSNs and mislocalization of proteins normally enriched in cilia. To visualize altered protein localization in OSNs, we generated a SLP3(eGFP) knock-in mouse and imaged the apical epithelium, including dendritic knobs and proximal cilia, in ex vivo tissue preparations. Additionally, protein reagents that reflect the characteristic neuronal activity of each OSN revealed altered activity in Bbs8-null cells. In addition to previously known defects at the ciliary border, we also observed aberrant targeting of OSN axons to the olfactory bulb; axons expressing the same receptor display reduced fasciculation and project to multiple targets in the olfactory bulb. We suggest that loss of BBS8 leads to a dramatic and variable reduction in cilia, the essential signaling platform for olfaction, which alters the uniformity of responses in populations of OSNs expressing the same receptor, thereby contributing to the observed axon-targeting defects.
Asunto(s)
Axones/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Trastornos del Olfato/fisiopatología , Proteínas/metabolismo , Olfato/fisiología , Animales , Síndrome de Bardet-Biedl/fisiopatología , Cilios/metabolismo , Proteínas del Citoesqueleto , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Mucosa Olfatoria/citología , Mucosa Olfatoria/fisiología , Proteínas/genética , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/fisiologíaRESUMEN
BACKGROUND: Chronic rhinosinusitis (CRS) is among the most common causes of olfactory loss. The loss of the sense of smell is thought to result from structural and functional changes occurring in the olfactory epithelium caused by inflammation. However, the cellular mechanisms underlying CRS-associated olfactory loss remain incompletely understood. METHODS: Transgenic mice expressing TNF-alpha specifically within the olfactory epithelium were used as a model for CRS-associated olfactory loss. TNF-alpha expression was induced over different time intervals, and olfactory epithelial tissue was assessed for the expression of neuronal markers by laser scanning confocal microscopy and Western blot. RESULTS: TNF-alpha expression results in an inflammatory infiltrate in the olfactory epithelium, thinning of the olfactory neuron layer, and a progressive loss of olfactory function. Reduced expression of markers for neurons and mature olfactory neurons (neural cell adhesion molecule [NCAM] and olfactory marker protein [OMP], respectively) was observed in the neuroepithelium and in the subepithelial axon bundles. Expression of growth-associated protein (GAP) 43, a marker for immature neurons, was also reduced. These alterations were reversed when TNF-alpha expression was discontinued. CONCLUSION: TNF-alpha expression in a transgenic model of CRS-associated olfactory loss results in progressive loss of olfactory neurons. Decreased GAP-43 expression suggests that TNF-alpha-associated inflammation inhibits differentiation of progenitor cells into immature olfactory neurons. Therefore, reduced regeneration of olfactory neurons may be an important mechanism underlying olfactory loss in CRS, in addition to neuronal loss or apoptosis. This mouse model represents a potential tool in the development of novel therapeutic strategies for the prevention of olfactory neuron loss in CRS.
Asunto(s)
Agnosia/metabolismo , Neuronas/metabolismo , Rinitis/metabolismo , Sinusitis/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Agnosia/etiología , Agnosia/genética , Agnosia/patología , Agnosia/fisiopatología , Animales , Antígenos de Diferenciación/metabolismo , Apoptosis , Enfermedad Crónica , Clonación Molecular , Proteína GAP-43/metabolismo , Inflamación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/patología , Proteína Marcadora Olfativa/metabolismo , Rinitis/complicaciones , Rinitis/genética , Rinitis/patología , Rinitis/fisiopatología , Sinusitis/complicaciones , Sinusitis/genética , Sinusitis/patología , Sinusitis/fisiopatología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Body fluids such as urine potentially contain a wealth of information pertaining to age, sex, social and reproductive status, physiologic state, and genotype of the donor. To explore whether urine could encode information regarding environment, physiology, and development, we compared the volatile compositions of mouse urine using solid-phase microextraction and gas chromatography-mass spectrometry (SPME-GC/MS). Specifically, we identified volatile organic compounds (VOCs) in individual urine samples taken from inbred C57BL/6J-H-2(b) mice under several experimental conditions-maturation state, diet, stress, and diurnal rhythms, designed to mimic natural variations. Approximately 1000 peaks (i.e., variables) were identified per comparison and of these many were identified as potential differential biomarkers. Consistent with previous findings, we found groups of compounds that vary significantly and consistently rather than a single unique compound to provide a robust signature. We identified over 49 new predictive compounds, in addition to identifying several published compounds, for maturation state, diet, stress, and time-of-day. We found a considerable degree of overlap in the chemicals identified as (potential) biomarkers for each comparison. Chemometric methods indicate that the strong group-related patterns in VOCs provide sufficient information to identify several parameters of natural variations in this strain of mice including their maturation state, stress level, and diet.
Asunto(s)
Biomarcadores/orina , Ritmo Circadiano/fisiología , Dieta , Maduración Sexual , Estrés Fisiológico , Animales , Cromatografía de Gases y Espectrometría de Masas , Masculino , Ratones , Ratones Endogámicos C57BL , Método de Montecarlo , Análisis de Componente Principal , Microextracción en Fase Sólida , Compuestos Orgánicos Volátiles/química , Compuestos Orgánicos Volátiles/aislamiento & purificación , Compuestos Orgánicos Volátiles/orinaRESUMEN
The worldwide epidemic of obesity has increased the urgency to develop a deeper understanding of physiological systems related to energy balance and energy storage, including the mechanisms controlling the development of fat cells (adipocytes). The differentiation of committed preadipocytes to adipocytes is controlled by PPARgamma and several other transcription factors, but the molecular basis for preadipocyte determination is not understood. Using a new method for the quantitative analysis of transcriptional components, we identified the zinc-finger protein Zfp423 as a factor enriched in preadipose versus non-preadipose fibroblasts. Ectopic expression of Zfp423 in non-adipogenic NIH 3T3 fibroblasts robustly activates expression of Pparg in undifferentiated cells and permits cells to undergo adipocyte differentiation under permissive conditions. Short hairpin RNA (shRNA)-mediated reduction of Zfp423 expression in 3T3-L1 cells blunts preadipocyte Pparg expression and diminishes the ability of these cells to differentiate. Furthermore, both brown and white adipocyte differentiation is markedly impaired in Zfp423-deficient mouse embryos. Zfp423 regulates Pparg expression, in part, through amplification of the BMP signalling pathway, an effect dependent on the SMAD-binding capacity of Zfp423. This study identifies Zfp423 as a transcriptional regulator of preadipocyte determination.
Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/metabolismo , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , PPAR gamma/metabolismo , Estructura Terciaria de Proteína , Proteínas Smad/metabolismoRESUMEN
Odorant-evoked activity contributes to olfactory epithelium organization and axon targeting. We examined the consequences on gene expression of a genetic disruption of the channel responsible for olfactory transduction. Genes encoding calcium-binding EF-hand motifs, were among the most highly regulated transcripts consistent with the central role of Ca(2+) influx in neuronal depolarization. Several genes encoding integral membrane proteins are also highly regulated. One gene, Lrrc3b, was regulated more than 10-fold by odorant activity. Changes in expression occur within thirty minutes and are maintained for several hours. In genetic disruptions of Lrrc3b, a Lrrc3b-promoter-driven reporter adopts the activity-regulated expression of the endogenous gene. Individual olfactory glomeruli have a wide spectrum of activity levels that can be modulated by altering odor exposure. The Lrrc3b reporter mouse permits direct assessment of activity in identified glomeruli. In stable odorant environments, activity-regulated proteins provide a characteristic signature that is correlated with the olfactory receptor they express.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Neuronas Receptoras Olfatorias/metabolismo , Olfato/genética , Animales , Northern Blotting , Western Blotting , Línea Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Humanos , Hibridación in Situ , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Odorantes , Neuronas Receptoras Olfatorias/fisiología , Regiones Promotoras Genéticas , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Olfato/fisiologíaRESUMEN
The paper discusses variable selection as used in large metabolomic studies, exemplified by mouse urinary gas chromatography of 441 mice in three experiments to detect the influence of age, diet, and stress on their chemosignal. Partial least squares discriminant analysis (PLS-DA) was applied to obtain class models, using a procedure of 20,000 iterations including the bootstrap for model optimization and random splits into test and training sets for validation. Variables are selected using PLS regression coefficients on the training set using an optimized number of components obtained from the bootstrap. The variables are ranked in order of significance, and the overall optimal variables are selected as those that appear as highly significant over 100 different test and training set splits. Cost/benefit analysis of performing the model on a reduced number of variables is also illustrated. This paper provides a strategy for properly validated methods for determining which variables are most significant for discriminating between two groups in large metabolomic data sets avoiding the common pitfall of overfitting if variables are selected on a combined training and test set and also taking into account that different variables may be selected each time the samples are split into training and test sets using iterative procedures.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Metabolómica/métodos , Animales , Área Bajo la Curva , Análisis Discriminante , Análisis de los Mínimos Cuadrados , Metaboloma , Metabolómica/economía , Ratones , Modelos Estadísticos , Modelos Teóricos , Urinálisis/economíaRESUMEN
unc-3 encodes the Caenorhabditis elegans ortholog of the Olf-1/Early B cell factor family of transcription factors, which in vertebrates regulate development and differentiation of B lymphocytes, adipocytes, and cells of the nervous system. unc-3 mutants are uncoordinated in locomotion. Here we show that unc-3 represses a VC-like motor neuron program in the VA and VB motor neurons, which in wild-type animals control backwards and forwards locomotion, respectively. We identify a physical interaction between UNC-3 and the C2H2 zinc finger transcription factor PAG-3, the mammalian homologs of which are coexpressed in olfactory epithelium and hematopoietic cells. Our data explain the locomotory defects of unc-3 mutants and suggest that interactions between unc-3 and pag-3 orthologs in other species may be functionally important.