RESUMEN
The transcription factor Sip1 (Zeb2) plays multiple roles during CNS development from early acquisition of neural fate to cortical neurogenesis and gliogenesis. In humans, SIP1 (ZEB2) haploinsufficiency leads to Mowat-Wilson syndrome, a complex congenital anomaly including intellectual disability, epilepsy and Hirschsprung disease. Here we uncover the role of Sip1 in retinogenesis. Somatic deletion of Sip1 from mouse retinal progenitors primarily affects the generation of inner nuclear layer cell types, resulting in complete loss of horizontal cells and reduced numbers of amacrine and bipolar cells, while the number of Muller glia is increased. Molecular analysis places Sip1 downstream of the eye field transcription factor Pax6 and upstream of Ptf1a in the gene network required for generating the horizontal and amacrine lineages. Intriguingly, characterization of differentiation dynamics reveals that Sip1 has a role in promoting the timely differentiation of retinal interneurons, assuring generation of the proper number of the diverse neuronal and glial cell subtypes that constitute the functional retina in mammals.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Retina/citología , Retina/metabolismo , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Linaje de la Célula , Inmunoprecipitación de Cromatina , Femenino , Técnica del Anticuerpo Fluorescente , Ratones , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Neurogénesis/fisiología , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Embarazo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
MgcRacGAP (RACGAP1) is a GTPase Activating Protein (GAP), highly produced in the mouse embryonic brain and in the human and mouse post-natal testis. MgcRacGAP negatively controls the activity of Rac and Cdc42, which are key molecular switches acting on the microtubule and actin cytoskeleton and controlling various cell processes such as proliferation, adhesion and motility. Previous studies demonstrated that MgcRacGAP plays a critical role in the cytokinesis of somatic cells; hence homozygous inactivation of the gene in the mouse and mutation in Caenorhabditis elegans led to embryonic lethality due to the inability of MgcRacGAP-null embryos to assemble the central spindle and to complete cytokinesis. In the testis, the germ cells do not complete cytokinesis and remain connected as a syncytium throughout the entire process of spermatogenesis. Interestingly, MgcRacGAP was shown to locate to the intercellular bridges, connecting these germ cells. In order to determine the function(s) of MgcRacGAP in the male germline, we generated a conditional knock-out mouse using Stra8 promoter driven Cre recombinase to induce the specific deletion of MgcRacGAP in the pre-meiotic germ cells. We found that the absence of MgcRacGAP induced a germline depletion and male sterility. Consistent with the role of MgcRacGAP in the establishment of the cytoplasm constriction during cytokinesis of the somatic cells, we observed that MgcRacGAP deletion in the germ cells prevented the formation of the intercellular bridges and induced a proliferation arrest. While we assume that inherited homozygous loss of function mutations in MgcRacGAP would be lethal in human, de novo mutations in the testis might account for some cases of non-obstructive oligo- and/or azoo-spermia syndromes, whose genetic causes are altogether still poorly defined.
Asunto(s)
Proteínas Activadoras de GTPasa/deficiencia , Células Germinativas/química , Infertilidad Masculina/genética , Espermatogénesis/fisiología , Testículo/metabolismo , Animales , Western Blotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Transgénicos , Microscopía Electrónica , Espermatogénesis/genética , Testículo/ultraestructuraRESUMEN
The involvement of SOX10 and ZFHX1B in Waardenburg-Hirschsprung disease (hypopigmentation, deafness, and absence of enteric ganglia) and Mowat-Wilson syndrome (mental retardation, facial dysmorphy and variable congenital malformations including Hirschsprung disease) respectively, highlighted the importance of both transcription factors during enteric nervous system (ENS) development. The expression and function of SOX10 are now well established, but those of ZFHX1B remain elusive. Here we describe the expression profile of Zfhx1b and its genetic interactions with Sox10 during mouse ENS development. Through phenotype analysis of Sox10;Zfhx1b double mutants, we show that a coordinated and balanced interaction between these two genes is required for normal ENS development. Double mutants present with more severe ENS defects due to decreased proliferation of enteric progenitors and increased neuronal differentiation from E11.5 onwards. Thus, joint activity between these two transcription factors is crucial for proper ENS development and our results contribute to the understanding of the molecular basis of ENS defects observed both in mutant mouse models and in patients carrying SOX10 and ZFHX1B mutations.
Asunto(s)
Sistema Nervioso Entérico/embriología , Proteínas de Homeodominio/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción SOXE/metabolismo , Animales , Cruzamientos Genéticos , Embrión de Mamíferos/metabolismo , Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Enfermedad de Hirschsprung/metabolismo , Proteínas de Homeodominio/genética , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Mutación , Neuroglía/metabolismo , Proteínas Represoras/genética , Factores de Transcripción SOXE/genética , Células Madre/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
OBJECTIVES: Osteoarthritis (OA) is the most prevalent form of arthritis and accounts for substantial morbidity and disability, particularly in older people. It is characterised by changes in joint structure, including degeneration of the articular cartilage, and its aetiology is multifactorial with a strong postulated genetic component. METHODS: A meta-analysis was performed of four genome-wide association (GWA) studies of 2371 cases of knee OA and 35 909 controls in Caucasian populations. Replication of the top hits was attempted with data from 10 additional replication datasets. RESULTS: With a cumulative sample size of 6709 cases and 44 439 controls, one genome-wide significant locus was identified on chromosome 7q22 for knee OA (rs4730250, p=9.2 × 10â»9), thereby confirming its role as a susceptibility locus for OA. CONCLUSION: The associated signal is located within a large (500 kb) linkage disequilibrium block that contains six genes: PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, ß), HPB1 (HMG-box transcription factor 1), COG5 (component of oligomeric golgi complex 5), GPR22 (G protein-coupled receptor 22), DUS4L (dihydrouridine synthase 4-like) and BCAP29 (B cell receptor-associated protein 29). Gene expression analyses of the (six) genes in primary cells derived from different joint tissues confirmed expression of all the genes in the joint environment.
Asunto(s)
Cromosomas Humanos Par 7/genética , Predisposición Genética a la Enfermedad , Osteoartritis de la Rodilla/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
Mutations in ZFHX1B cause Mowat-Wilson syndrome (MWS) but the precise mechanisms underlying the aberrant functions of mutant ZFHX1B proteins (also named Smad-interacting protein-1, SIP1) in patients are unknown. Using mass spectrometry analysis, we identified subunits of the NuRD corepressor complex in affinity-purified Zfhx1b complexes. We find that Zfhx1b associates with NuRD through its N-terminal domain, which contains a previously postulated NuRD interacting motif. Interestingly, this motif is substituted by an unrelated sequence in a recently described MWS patient. We show here that such aberrant ZFHX1B protein is unable to recruit NuRD subunits and displays reduced transcriptional repression activity on the XBMP4 gene promoter, a target of Zfhx1b. We further demonstrate that the NuRD component Mi-2beta is involved in repression of the Zfhx1b target gene E-cadherin as well as in Zfhx1b-induced neural induction in animal caps from Xenopus embryos. Thus, NuRD and Zfhx1b functionally interact, and defective NuRD recruitment by mutant human ZFHX1B can be a MWS-causing mechanism. This is the first study providing mechanistic insight into the aberrant function of a single domain of the multi-domain protein ZFHX1B/SIP1 in human disease.
Asunto(s)
Anomalías Múltiples/metabolismo , Histona Desacetilasas/metabolismo , Discapacidad Intelectual/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Autoantígenos/metabolismo , Cadherinas/metabolismo , Línea Celular , ADN Helicasas/metabolismo , Embrión no Mamífero/metabolismo , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Síndrome , XenopusRESUMEN
BACKGROUND: Kabuki syndrome (KS) is a rare, clinically recognisable, congenital mental retardation syndrome. The aetiology of KS remains unknown. METHODS: Four carefully selected patients with KS were screened for chromosomal imbalances using array comparative genomic hybridisation at 1 Mb resolution. RESULTS: In one patient, a 250 kb de novo microdeletion at 20p12.1 was detected, deleting exon 5 of C20orf133. The function of this gene is unknown. In situ hybridisation with the mouse orthologue of C20orf133 showed expression mainly in brain, but also in kidney, eye, inner ear, ganglia of the peripheral nervous system and lung. CONCLUSION: The de novo nature of the deletion, the expression data and the fact that C20orf133 carries a macro domain, suggesting a role for the gene in chromatin biology, make the gene a likely candidate to cause the phenotype in this patient with KS. Both the finding of different of chromosomal rearrangements in patients with KS features and the absence of C20orf133 mutations in 19 additional patients with KS suggest that KS is genetically heterogeneous.
Asunto(s)
Anomalías Múltiples/genética , Cromosomas Humanos Par 20/genética , Discapacidad Intelectual/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Cromosomas Humanos Par 20/química , Cromosomas Humanos Par 20/ultraestructura , Enzimas Reparadoras del ADN , Exones/genética , Cara/anomalías , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Hidrolasas , Recién Nacido , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Especificidad de Órganos , Fenotipo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Síndrome , Factores de Transcripción/deficiencia , Factores de Transcripción/fisiologíaRESUMEN
OBJECTIVE: One of the early hallmarks of osteoarthritis (OA) is a progressive degeneration of the articular cartilage. Early diagnosis of OA-associated cartilage alterations would be beneficial for disease prevention and control, and for the development of disease-modifying treatments. However, early diagnosis is still hampered by a lack of quantifiable readouts in preclinical models. DESIGN: In this study, we have shown the potency of contrast-enhanced nanofocus x-ray computed tomography (CE-nanoCT) to be used for virtual 3-dimensional (3D) histopathology in established mouse models for OA, and we compared with standard histopathology. RESULTS: We showed the equivalence of CE-nanoCT images to histopathology for the modified Mankin scoring of the cartilage structure and quality. Additionally, a limited set of 3D cartilage characteristics measured by CE-nanoCT image analysis in a user-independent and semiautomatic manner, that is, average and maximum of the noncalcified cartilage thickness distribution and loss in glycosaminoglycans, was shown to be predictive for the cartilage quality and structure as can be evaluated by histopathological scoring through the use of an empirical model. CONCLUSIONS: We have shown that CE-nanoCT is a tool that allows virtual histopathology and 3D morphological quantification of multitissue systems, such as the chondro-osseous junction. It provides faster and more quantitative data on cartilage structure and quality compared with standard histopathology while eliminating user bias. CE-nanoCT thus should allow capturing subtle differences in cartilage characteristics, carefully mapping OA progression and, ultimately, asses the beneficial changes when testing a candidate disease-modifying treatment.
RESUMEN
Bioluminescence imaging assays have become a widely integrated technique to quantify effectiveness of cell-based therapies by monitoring fate and survival of transplanted cells. To date these assays are still largely qualitative and often erroneous due to the complexity and dynamics of local micro-environments (niches) in which the cells reside. Here, we report, using a combined experimental and computational approach, on oxygen that besides being a critical niche component responsible for cellular energy metabolism and cell-fate commitment, also serves a primary role in regulating bioluminescent light kinetics. We demonstrate the potential of an oxygen dependent Michaelis-Menten relation in quantifying intrinsic bioluminescence intensities by resolving cell-associated oxygen gradients from bioluminescent light that is emitted from three-dimensional (3D) cell-seeded hydrogels. Furthermore, the experimental and computational data indicate a strong causal relation of oxygen concentration with emitted bioluminescence intensities. Altogether our approach demonstrates the importance of oxygen to evolve towards quantitative bioluminescence and holds great potential for future microscale measurement of oxygen tension in an easily accessible manner.
Asunto(s)
Oxígeno/química , Oxígeno/metabolismo , Línea Celular , Humanos , Hidrogeles , Mediciones LuminiscentesRESUMEN
By providing a three-dimensional (3D) support to cells, hydrogels offer a more relevant in vivo tissue-like environment as compared to two-dimensional cell cultures. Hydrogels can be applied as screening platforms to investigate in 3D the role of biochemical and biophysical cues on cell behaviour using bioluminescent reporter cells. Gradients in oxygen concentration that result from the interplay between molecular transport and cell metabolism can however cause substantial variability in the observed bioluminescent reporter cell activity. To assess the influence of these oxygen gradients on the emitted bioluminescence for various hydrogel geometries, a combined experimental and modelling approach was implemented. We show that the applied model is able to predict oxygen gradient independent bioluminescent intensities which correlate better to the experimentally determined viable cell numbers, as compared to the experimentally measured bioluminescent intensities. By analysis of the bioluminescence reaction dynamics we obtained a quantitative description of cellular oxygen metabolism within the hydrogel, which was validated by direct measurements of oxygen concentration within the hydrogel. Bioluminescence peak intensities can therefore be used as a quantitative measurement of reporter cell activity within a hydrogel, but an unambiguous interpretation of these intensities requires a compensation for the influence of cell-induced oxygen gradients on the luciferase activity.
Asunto(s)
Hidrogeles/química , Mediciones Luminiscentes/métodos , Oxígeno/química , Recuento de Células , Supervivencia Celular , Células Cultivadas , Recuperación de Fluorescencia tras Fotoblanqueo , Genes Reporteros , Células HEK293 , Humanos , Imagenología Tridimensional , Lentivirus/genética , Luminiscencia , Modelos Teóricos , SefarosaRESUMEN
OBJECTIVE: Autologous chondrocyte implantation (ACI) involves the application of a chondrocyte suspension into a membrane-sealed cartilage defect. Recently, "cell-seeded collagen matrix-supported" ACI has been developed wherein chondrocytes are seeded on a biomembrane. This study aimed at preclinically comparing 4 variant ACI techniques in a refined goat model: 2 traditional procedures, whereby the defect is sealed by a periosteal flap or collagen membrane, and 2 cell-seeding methods, with the collagen membrane either sutured or glued into the defect. DESIGN: The efficacy of the surgical techniques was evaluated in an acute critical size chondral defect in the medial condyle of 32 skeletally mature goats, randomly assigned to 1 of the 4 aforementioned treatment groups. After 10 weeks in vivo, the quality of the repair was graded histologically by 2 independent, blinded readers using the "modified O'Driscoll" score. RESULTS: The cell-seeding procedure whereby the membrane is sutured into the defect has a similar structural repair capacity than traditional ACI techniques. However, when the cell-seeded membrane was glued into the defect, the outcome appeared inferior. CONCLUSION: These findings indicate that optimizing the goat model and the postoperative recovery does allow preclinical evaluation of ACI-based cartilage implants in a load-bearing setting. This preclinical observation provides support to the clinical utilization of the sutured membrane-seeded (ACI-CS) technique, provided sutures, but not fibrin sealants, are used to fix the cell-seeded membrane in the defect bed.
RESUMEN
The magnetic properties of grain-oriented (GO) electrical steels strongly depend on the distribution of the α and ß angles, i.e., the deviations of the easy magnetisation <100> from the rolling direction (RD) in the rolling plane and out of the rolling plane, respectively. However, most Electron Backscatter Diffraction (EBSD) studies consider the standard Goss deviation angle, which includes the rotation of the (110) plane about the RD. Therefore, in the present work, a new procedure is demonstrated for deriving the α and ß angles from EBSD mappings to obtain a quantitative texture characterisation in line with the magnetic properties. This procedure is later applied to 37 GO steels after secondary recrystallisation that exhibit a wide range of permeability levels. The relation between the texture and the polarisation at 800A/m (J800) that is measured in the present study by EBSD is compared to the one that has been determined in previous papers with optical goniometers and X-ray diffraction techniques, and this relation is subsequently used to define a relevant parameter to describe the orientation quality of the grains. The results indicate that the average angle of the α and ß deviations is a relevant deviation parameter for the characterisation of grain orientations. Finally, it is demonstrated that the combination of the quantitative correlation between polarisation and texture with the orientation imaging of EBSD offers the possibilities of both studying the crystallographic environment of highly oriented grains in the primary recrystallised matrix for the production of high-permeability steels and evaluating the spatial distribution of the angular deviations in GO steels after secondary recrystallisation.
RESUMEN
Molecular oxygen is a main regulator of various cell functions. Imaging methods designed as screening tools for fast, in situ, 3D and non-interfering measurement of oxygen tension in the cellular microenvironment would serve great purpose in identifying and monitoring this vital and pivotal signalling molecule. We describe the use of dual luminophore oxygen sensitive microbeads to measure absolute oxygen concentrations in cellular aggregates. Stable microbead integration, a prerequisite for their practical application, was ensured by a site-specific delivery method that is based on the interactions between streptavidin and biotin. The spatial stability introduced by this method allowed for long term measurements of oxygen tension without interfering with the cell aggregation process. By making multiple calibration experiments we further demonstrated the potential of these sensors to measure local oxygen tension in optically dense cellular environments.
Asunto(s)
Bioensayo/métodos , Condrocitos/metabolismo , Imagen Molecular/métodos , Oximetría/métodos , Consumo de Oxígeno/fisiología , Oxígeno/análisis , Espectrometría de Fluorescencia/métodos , Animales , Agregación Celular/fisiología , Línea Celular , Condrocitos/citología , Ratones , MicroesferasRESUMEN
Members of the Nuclear eXport Factor (NXF) family are involved in the export of mRNA from the nucleus to the cytoplasm, or hypothesized to play a role in transport of cytoplasmic mRNA. We previously reported on the loss of NXF5 in a male patient with a syndromic form of intellectual disability. To study the functional role of NXF5 we identified the mouse counterpart. Based on synteny, mouse Nxf2 is the ortholog of human NXF5. However, we provide several lines of evidence that mouse Nxf7 is the actual functional equivalent of NXF5. Both Nxf7 and NXF5 are predominantly expressed in the brain, show cytoplasmic localization, and present as granules in neuronal dendrites suggesting a role in cytoplasmic mRNA metabolism in neurons. Nxf7 was primarily detected in the pyramidal cells of the hippocampus and in layer V of the cortex. Similar to human NXF2, mouse Nxf2 is highly expressed in testis and shows a nuclear localization. Interestingly, these findings point to a different evolutionary path for both NXF genes in human and mouse. We thus generated and validated Nxf7 knockout mice, which were fertile and did not present any gross anatomical or morphological abnormalities. Expression profiling in the hippocampus and the cortex did not reveal significant changes between wild-type and Nxf7 knockout mice. However, impaired spatial memory was observed in these KO mice when evaluated in the Morris water maze test. In conclusion, our findings provide strong evidence that mouse Nxf7 is the functional counterpart of human NXF5, which might play a critical role in mRNA metabolism in the brain.
Asunto(s)
Corteza Cerebral/metabolismo , Efecto Fundador , Hipocampo/metabolismo , Discapacidad Intelectual/genética , Neuronas/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Unión al ARN/genética , Transporte Activo de Núcleo Celular/genética , Animales , Corteza Cerebral/patología , Corteza Cerebral/ultraestructura , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Modelos Animales de Enfermedad , Expresión Génica , Hipocampo/patología , Hipocampo/ultraestructura , Humanos , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Masculino , Aprendizaje por Laberinto , Memoria , Ratones , Ratones Noqueados , Neuronas/patología , Neuronas/ultraestructura , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Especificidad de Órganos , Proteínas de Unión al ARN/metabolismo , Testículo/metabolismoRESUMEN
OBJECTIVE: To identify novel genes involved in osteoarthritis (OA), by means of a genome-wide association study. METHODS: We tested 500,510 single-nucleotide polymorphisms (SNPs) in 1,341 Dutch Caucasian OA cases and 3,496 Dutch Caucasian controls. SNPs associated with at least 2 OA phenotypes were analyzed in 14,938 OA cases and approximately 39,000 controls. Meta-analyses were performed using the program Comprehensive Meta-analysis, with P values <1 x 10(-7) considered genome-wide significant. RESULTS: The C allele of rs3815148 on chromosome 7q22 (minor allele frequency 23%; intron 12 of the COG5 gene) was associated with a 1.14-fold increased risk (95% confidence interval 1.09-1.19) of knee and/or hand OA (P = 8 x 10(-8)) and also with a 30% increased risk of knee OA progression (95% confidence interval 1.03-1.64) (P = 0.03). This SNP is in almost complete linkage disequilibrium with rs3757713 (68 kb upstream of GPR22), which is associated with GPR22 expression levels in lymphoblast cell lines (P = 4 x 10(-12)). Immunohistochemistry experiments revealed that G protein-coupled receptor protein 22 (GPR22) was absent in normal mouse articular cartilage or synovium. However, GPR22-positive chondrocytes were found in the upper layers of the articular cartilage of mouse knee joints that were challenged with in vivo papain treatment or methylated bovine serum albumin treatment. GPR22-positive chondrocyte-like cells were also found in osteophytes in instability-induced OA. CONCLUSION: Our findings identify a novel common variant on chromosome 7q22 that influences susceptibility to prevalence and progression of OA. Since the GPR22 gene encodes a G protein-coupled receptor, this is potentially an interesting therapeutic target.
Asunto(s)
Cromosomas Humanos Par 7 , Estudio de Asociación del Genoma Completo , Osteoartritis de la Cadera/genética , Osteoartritis de la Rodilla/genética , Población Blanca/genética , Animales , Cartílago Articular/efectos de los fármacos , Cartílago Articular/patología , Cartílago Articular/fisiología , Línea Celular , Femenino , Predisposición Genética a la Enfermedad , Humanos , Linfocitos/citología , Linfocitos/fisiología , Masculino , Ratones , Países Bajos , Osteoartritis de la Cadera/etnología , Osteoartritis de la Rodilla/etnología , Papaína/farmacología , Fenotipo , Polimorfismo de Nucleótido Simple , Prevalencia , Receptores Acoplados a Proteínas G/genética , Factores de Riesgo , Albúmina Sérica Bovina/farmacología , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/patología , Membrana Sinovial/fisiología , Población Blanca/estadística & datos numéricosRESUMEN
Gene trapping in mouse embryonic stem (ES) cells enables near-saturation vector-based insertional mutagenesis across the genome of this model organism. About 135,000 trapped ES cell lines are made available to the scientific community by the International Gene Trap Consortium (IGTC; www.genetrap.org). A search of one of its databases identified an ES cell line (RRS512) with a betaGeo-based gene trap (gt) vector insertion in intron 5 of Ttrap, a gene that encodes an intracellular signalling protein, which is implicated in gastrulation movement and left-right asymmetry in zebrafish embryos. We have determined the exact gt insertion point in the mutant ES cell clone RRS512 and confirmed the production of a chimaeric transcript consisting of the upstream Ttrap exons and the gene trap vector encoded marker/selection fusion sequences. This ES cell line was used to generate heterozygous Ttrap mutant mice, which were further crossed to obtain Ttrap(gt/gt) mice. In contrast to Ttraps documented essential role during nodal and Smad3 controlled zebrafish early embryogenesis, Ttrap(gt/gt) mice were born with a normal Mendelian distribution. However, subsequent analysis of these Ttrap(gt/gt) mice has revealed a duplication of the wild-type Ttrap allele that was already present in the RRS512 cell line. Based on our detailed analysis presented here, we suggest an extensive procedure for the characterization of gene trap ES cell lines prior to generating gene trap mice with these.
Asunto(s)
Células Madre Embrionarias/metabolismo , Vectores Genéticos , Mutagénesis Insercional/métodos , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Femenino , Técnicas Genéticas , Hibridación in Situ , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Kabuki syndrome (KS) is a rare, congenital mental retardation syndrome. The aetiology of KS remains unknown. Four carefully selected patients with KS were screened for chromosomal imbalances using array comparative genomic hybridisation at 1 Mb resolution. In one patient, a 250 kb de novo microdeletion at 20p12.1 was detected, deleting exon 5 of C20orf133. The function of this gene is unknown. In situ hybridisation with the mouse orthologue of C20orf133 showed expression mainly in brain. The de novo nature of the deletion, the expression data and the fact that C20orf133 carries a macro domain, suggesting a role for the gene in chromatin biology, make the gene a likely candidate to cause the phenotype in this patient with KS. Both the finding of different of chromosomal rearrangements in patients with KS features and the absence of C20orf133 mutations in 19 additional patients with KS suggest that KS is genetically heterogeneous.
RESUMEN
Mowat-Wilson syndrome is a recently delineated autosomal dominant developmental anomaly, whereby heterozygous mutations in the ZFHX1B gene cause mental retardation, delayed motor development, epilepsy and a wide spectrum of clinically heterogeneous features, suggestive of neurocristopathies at the cephalic, cardiac and vagal levels. However, our understanding of the etiology of this condition at the cellular level remains vague. This study presents the Zfhx1b protein expression domain in mouse embryos and correlates this with a novel mouse model involving a conditional mutation in the Zfhx1b gene in neural crest precursor cells. These mutant mice display craniofacial and gastrointestinal malformations that show resemblance to those found in human patients with Mowat-Wilson syndrome. In addition to these clinically recognized alterations, we document developmental defects in the heart, melanoblasts and sympathetic and parasympathetic anlagen. The latter observations in our mouse model for Mowat-Wilson suggest a hitherto unknown role for Zfhx1b in the development of these particular neural crest derivatives, which is a set of observations that should be acknowledged in the clinical management of this genetic disorder.
Asunto(s)
Anomalías Múltiples/genética , Proteínas de Homeodominio/genética , Cresta Neural/embriología , Proteínas Represoras/genética , Anomalías Múltiples/embriología , Animales , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/metabolismo , Femenino , Ganglios Sensoriales/embriología , Ganglios Sensoriales/metabolismo , Tracto Gastrointestinal/embriología , Tracto Gastrointestinal/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Masculino , Melanocitos/metabolismo , Ratones , Ratones Noqueados , Mutación , Cresta Neural/anomalías , Cresta Neural/metabolismo , Proteínas Represoras/metabolismo , Cráneo/embriología , Cráneo/metabolismo , Sistema Nervioso Simpático/embriología , Sistema Nervioso Simpático/metabolismo , Síndrome , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
Smad-interacting protein-1 (Sip1) [Zinc finger homeobox (Zfhx1b)] is a transcription factor implicated in the genesis of Mowat-Wilson syndrome in humans. Sip1 expression in the dorsal telencephalon of mouse embryos was documented from E12.5. We inactivated the gene specifically in cortical precursors. This resulted in the lack of the entire hippocampal formation. Sip1 mutant mice exhibited death of differentiating cells and decreased proliferation in the region of the prospective hippocampus and dentate gyrus. The expression of the Wnt antagonist Sfrp1 was ectopically activated, whereas the activity of the noncanonical Wnt effector, JNK, was down-regulated in the embryonic hippocampus of mutant mice. In cortical cells, Sip1 protein was detected on the promoter of Sfrp1 gene and both genes showed a mutually exclusive pattern of expression suggesting that Sfrp1 expression is negatively regulated by Sip1. Sip1 is therefore essential to the development of the hippocampus and dentate gyrus, and is able to modulate Wnt signaling in these regions.
Asunto(s)
Hipocampo/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Apoptosis , Proliferación Celular , Eliminación de Gen , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Hipocampo/citología , Hipocampo/embriología , Hipocampo/crecimiento & desarrollo , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mutación/genética , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Células Madre/citología , Células Madre/metabolismo , Telencéfalo/metabolismo , Regulación hacia Arriba , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
In mouse embryos, the Zfhx1 transcription factor genes, Sip1 and deltaEF1, are expressed in complementary domains in many tissues. Their possible synergism in embryogenesis was investigated by comparing the phenotype of Sip1-/-;deltaEF1-/- double homozygotes with single homozygous embryos. Unexpectedly, in Sip1-/- embryos deltaEF1 was ectopically activated, suggesting a negative regulation of deltaEF1 expression by Sip1. Sip1-/-;deltaEF1-/- embryos were similar to Sip1-/- embryos in short somite production and developmental arrest around E8.5, but showed more severe defects in dorsal neural tube morphogenesis accompanied by a larger reduction of Sox2 expression, ascribable to the loss of the ectopic deltaEF1 expression. Sip1+/-;deltaEF1-/- embryos develop various morphological defects after E10 that were absent in deltaEF1-/- embryos even in tissues without significant overlap of Sip1 and deltaEF1 expression, and arrested during mid gestation earlier than deltaEF1-/- embryos. These findings indicate that complex synergistic interactions occur between Zfhx1 transcription factor genes during mouse embryogenesis.