RESUMEN
In the spinal cord, the central canal forms through a poorly understood process termed dorsal collapse that involves attrition and remodelling of pseudostratified ventricular layer (VL) cells. Here, we use mouse and chick models to show that dorsal ventricular layer (dVL) cells adjacent to dorsal midline Nestin(+) radial glia (dmNes+RG) down-regulate apical polarity proteins, including Crumbs2 (CRB2) and delaminate in a stepwise manner; live imaging shows that as one cell delaminates, the next cell ratchets up, the dmNes+RG endfoot ratchets down, and the process repeats. We show that dmNes+RG secrete a factor that promotes loss of cell polarity and delamination. This activity is mimicked by a secreted variant of Crumbs2 (CRB2S) which is specifically expressed by dmNes+RG. In cultured MDCK cells, CRB2S associates with apical membranes and decreases cell cohesion. Analysis of Crb2F/F/Nestin-Cre+/- mice, and targeted reduction of Crb2/CRB2S in slice cultures reveal essential roles for transmembrane CRB2 (CRB2TM) and CRB2S on VL cells and dmNes+RG, respectively. We propose a model in which a CRB2S-CRB2TM interaction promotes the progressive attrition of the dVL without loss of overall VL integrity. This novel mechanism may operate more widely to promote orderly progenitor delamination.
Asunto(s)
Proteínas de la Membrana/metabolismo , Médula Espinal/citología , Médula Espinal/embriología , Animales , Adhesión Celular , Embrión de Pollo , Perros , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Uniones Estrechas/metabolismo , Imagen de Lapso de TiempoRESUMEN
In humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analysed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganization was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalization was detected throughout the CRB2-deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors and disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells. The number of late-born progenitor cells, rod photoreceptors and Müller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late-born retinal progenitor cells.
Asunto(s)
Proteínas del Ojo/genética , Proteínas de la Membrana/genética , Mutación , Proteínas del Tejido Nervioso/genética , Retina/metabolismo , Retinitis Pigmentosa/genética , Animales , Apoptosis , Secuencia de Bases , Cartilla de ADN , Electrorretinografía , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Fotorreceptoras de Vertebrados/patología , Reacción en Cadena de la Polimerasa , Retina/crecimiento & desarrollo , Retinitis Pigmentosa/patología , Tomografía de Coherencia ÓpticaRESUMEN
ESCs undergoing neural differentiation in vitro display an intrinsic heterogeneity with a large subset of the cells forming polarized neural rosettes that maintain the neural progenitor microenvironment. This heterogeneity is not only necessary for normal development but also causes substantial technical challenges for practical applications. Here, we report a novel regulator of early neural progenitors, the apical polarity protein Crb2 (Crumbs homologue 2). Employing monolayer differentiation of mouse ESCs to model neurogenesis in vitro, we find that Crb2 is upregulated with Sox1 and Musashi at the onset of neuroepithelial specification and localizes to the apical side of neural rosettes. Stable Crb2-knockdown (KD) lines die at the onset of neural specification and fail to stabilize several apical polarity proteins. However, these cells are able to proliferate under self-renewing conditions and can be differentiated into mesodermal and endodermal lineages. Conversely, Crb2 overexpression during neural differentiation results in elevated levels of other apical polarity proteins and increases proliferation. Additionally, sustained overexpression of Crb2 reduces terminal differentiation into TuJ1-positive neurons. Furthermore, we demonstrate that Crb2 overexpression under self-renewing conditions increases glycogen synthase kinase (GSK)-3ß inhibition, correlating with an increase in clonogenicity. To confirm the importance of GSK-3ß inhibition downstream of Crb2, we show that Crb2-KD cells can be forced into neural lineages by blocking GSK-3ß function and supplementing Epidermal Growth Factor (EGF) and basic Fibroblast Growth Factor (bFGF). Thus, this is the first demonstration that a member of the Crumbs family is essential for survival and differentiation of ESC-derived neural progenitors.
Asunto(s)
Polaridad Celular , Células Madre Embrionarias/metabolismo , Proteínas de la Membrana/biosíntesis , Células-Madre Neurales/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Madre Embrionarias/citología , Factor de Crecimiento Epidérmico/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Proteínas de la Membrana/genética , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/citología , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Formación de Roseta , Factores de Transcripción SOXB1/biosíntesis , Factores de Transcripción SOXB1/genética , Tubulina (Proteína)/biosíntesisRESUMEN
PURPOSE: Mutations in the Crumbs homolog 1 (CRB1) gene cause autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Database searches reveal two other Crumbs homologs on chromosomes 9q33.3 and 19p13.3. The purpose of this study was to characterize the Crumbs homolog 2 (CRB2) gene on 9q33.3, to analyze its expression pattern, and to determine whether mutations in CRB2 are associated with RP and LCA. METHODS: The CRB2 mRNA and its expression pattern in human tissues were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The cellular expression of Crb2 in the mouse eye was determined by mRNA in situ hybridizations. The open reading frame and splice junctions of CRB2 were analyzed for mutations by single-strand conformation analysis and direct nucleotide sequencing in 85 RP patients and 79 LCA patients. RESULTS: The CRB2 gene consists of 13 exons and encodes a 1285 amino acid transmembrane protein. CRB2 is mainly expressed in retina, brain, and kidney. In mouse retina Crb2 expression was detected in all cell layers. Mutation analysis of the CRB2 gene revealed 11 sequence variants leading to an amino acid substitution. Three of them were not identified in control individuals and affect conserved amino acid residues. However, the patients that carry these sequence variants do not have a second sequence variant on the other allele, excluding autosomal recessive inheritance of CRB2 sequence variants as a cause of their disease. CONCLUSIONS: This study shows that CRB2 sequence variants are not a common cause of autosomal recessive RP and LCA. It is possible that a more complex clinical phenotype is associated with the loss or altered function of CRB2 in humans due to its expression in tissues other than the retina.
Asunto(s)
Ceguera/genética , Proteínas Portadoras/genética , Cromosomas Humanos Par 9/genética , Proteínas de la Membrana/genética , Mutación , Retinitis Pigmentosa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ceguera/congénito , Encéfalo/metabolismo , Análisis Mutacional de ADN , Regulación de la Expresión Génica/fisiología , Variación Genética , Humanos , Hibridación in Situ , Riñón/metabolismo , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/metabolismo , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The mouse nude mutation inactivates the gene encoding the Foxn1 transcription factor, causing defective hair morphogenesis. Here, we show for the first time that Foxn1 is required for proper assembly of the hair medulla, and we identify Foxn1-regulated genes by transcript profiling. One such gene encodes the desmosomal cadherin, Dsc2. Significantly, Foxn1-dependent Dsc2 expression is restricted to the hair medulla, and within these cells, Dsc2 protein is predominantly localized to specialized adhesion junctions between the cortex and the medulla. Our results reveal Foxn1 as an essential regulator of tissue assembly in the growing hair shaft and implicate Dsc2 as a downstream effector of this activity.
Asunto(s)
Cadherinas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Desmosomas/fisiología , Regulación de la Expresión Génica/fisiología , Folículo Piloso/fisiología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Animales , Cadherinas/genética , Desmocolinas , Desmosomas/genética , Factores de Transcripción Forkhead , Regulación de la Expresión Génica/genética , Folículo Piloso/citología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones MutantesRESUMEN
Loss of Crumbs homologue 1 (CRB1) function causes either the eye disease Leber congenital amaurosis or progressive retinitis pigmentosa, depending on the amount of residual CRB1 activity and the genetic background. Crb1 localizes specifically to the sub-apical region adjacent to the adherens junction complex at the outer limiting membrane in the retina. We show that it is associated here with multiple PDZ protein 1 (Mupp1), protein associated with Lin-7 (Pals1 or Mpp5) and Mpp4. We have produced Crb1(-/-) mice completely lacking any functional Crb1. Although the retinas are initially normal, by 3-9 months the Crb1(-/-) retinas develop localized lesions where the integrity of the outer limiting membrane is lost and giant half rosettes are formed. After delamination of the photoreceptor layer, neuronal cell death occurs in the inner and outer nuclear layers of the retina. On moderate exposure to light for 3 days at 3 months of age, the number of severe focal retinal lesions significantly increases in the Crb1(-/-) retina. Crb2, Crb3 and Crb1 interacting proteins remain localized to the sub-apical region and therefore are not sufficient to maintain cell adhesion during light exposure in Crb1(-/-) retinas. Thus we propose that during light exposure Crb1 is essential to maintain, but not assemble, adherens junctions between photoreceptors and Müller glia cells and prevents retinal disorganization and dystrophy. Hence, light may be an influential factor in the development of the corresponding human diseases.
Asunto(s)
Polaridad Celular/fisiología , Luz , Proteínas del Tejido Nervioso/genética , Células Fotorreceptoras de Vertebrados/fisiología , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Degeneración Retiniana/metabolismo , Uniones Adherentes/metabolismo , Animales , Ceguera/genética , Ceguera/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Adhesión Celular/fisiología , Adhesión Celular/efectos de la radiación , Comunicación Celular/fisiología , Comunicación Celular/efectos de la radiación , Membrana Celular/metabolismo , Polaridad Celular/efectos de la radiación , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Estimulación Luminosa , Células Fotorreceptoras de Vertebrados/citología , Degeneración Retiniana/genética , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismoRESUMEN
MOTIVATION: Gene expression levels are obtained from microarray experiments through the extraction of pixel intensities from a scanned image of the slide. It is widely acknowledged that variabilities can occur in expression levels extracted from the same images by different users with the same software packages. These inconsistencies arise due to differences in the refinement of the placement of the microarray 'grids'. We introduce a novel automated approach to the refinement of grid placements that is based upon the use of Bayesian inference for determining the size, shape and positioning of the microarray 'spots', capturing uncertainty that can be passed to downstream analysis. RESULTS: Our experiments demonstrate that variability between users can be significantly reduced using the approach. The automated nature of the approach also saves hours of researchers' time normally spent in refining the grid placement.
Asunto(s)
Algoritmos , Artefactos , Perfilación de la Expresión Génica/métodos , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Fluorescente/métodos , Modelos Estadísticos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Teorema de Bayes , Variación Genética , Modelos Genéticos , Variaciones Dependientes del Observador , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Pax6 is a member of an evolutionarily conserved family of transcription factors. It is developmentally regulated and is required for the normal embryonic development of the central nervous system, eye and pancreas. Pax6 mutations in the mouse result in the Small eye (Sey) phenotype. Heterozygous mice have eye defects and homozygotes die immediately after birth lacking eyes, nasal cavities and with severe brain abnormalities, including a malformed cerebral cortex. Recent work has established that there are changes in expression of cell adhesion molecules and these may underlie at least a part of the Pax6(Sey/Sey) phenotype. Here we used cell transplants and explant cultures to investigate the role of Pax6 in cell adhesion. Pax6(Sey/Sey) embryonic cortical cells transplanted into wild-type embryonic cortex were observed to segregate from wild-type cells and form dense clusters. Cells migrating from explants of Pax6(Sey/Sey) embryonic cortex clustered to a greater extent than cells migrating from wild-type controls. These new data support the hypothesis that Pax6 exerts a cell-autonomous effect on the adhesiveness of cortical cells.
Asunto(s)
Adhesión Celular/fisiología , Corteza Cerebral/embriología , Proteínas de Homeodominio/fisiología , Neuronas/citología , Neuronas/fisiología , Animales , Animales Recién Nacidos , Diferenciación Celular , Corteza Cerebral/fisiología , Técnicas de Cultivo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/fisiología , Proteínas del Ojo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Mutantes Neurológicos , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Ratas , Proteínas Represoras , Células Madre/citología , Células Madre/fisiología , Trasplante de TejidosRESUMEN
In the proliferative zone of the developing cerebral cortex, multipotential progenitors predominate early in development and divide to increase the progenitor pool. As corticogenesis progresses, proportionately fewer progenitors are produced and, instead, cell divisions yield higher numbers of postmitotic neurones or glial cells. As the switch from the generation of progenitors to that of differentiated cells occurs, the orientation of cell division alters from predominantly symmetrical to predominantly asymmetrical. It has been hypothesised that symmetrical divisions expand the progenitor pool, whereas asymmetrical divisions generate postmitotic cells, although this remains to be proved. The molecular mechanisms regulating these processes are poorly understood. The transcription factor Pax6 is highly expressed in the cortical proliferative zone and there are morphological defects in the Pax6(Sey/Sey) (Pax6 null) cortex, but little is known about the principal cellular functions of Pax6 in this region. We have analysed the cell-cycle kinetics, the progenitor cleavage orientation and the onset of expression of differentiation markers in Pax6(Sey/Sey) cortical cells in vivo and in vitro. We showed that, early in corticogenesis at embryonic day (E) 12.5, the absence of Pax6 accelerated cortical development in vivo, shortening the cell cycle and the time taken for the onset of expression of neural-specific markers. This also occurred in dissociated culture of isolated cortical cells, indicating that the changes were intrinsic to the cortical cells. From E12.5 to E15.5, proportions of asymmetrical divisions increased more rapidly in mutant than in wild-type embryos. By E15.5, interkinetic nuclear migration during the cell cycle was disrupted and the length of the cell cycle was significantly longer than normal in the Pax6(Sey/Sey) cortex, with a lengthening of S phase. Together, these results show that Pax6 is required in developing cortical progenitors to control the cell-cycle duration, the rate of progression from symmetrical to asymmetrical division and the onset of expression of neural-specific markers.