RESUMEN
Primary cilia defects result in a group of related pleiotropic malformation syndromes known as ciliopathies, often characterised by cerebellar developmental and foliation defects. Here, we describe the cerebellar anatomical and signalling defects in the Tmem67tm1(Dgen)/H knockout mouse. At mid-gestation, Tmem67 mutant cerebella were hypoplastic and had aberrantly high canonical Wnt/ß-catenin signalling, proliferation and apoptosis. Later in development, mutant cerebellar hemispheres had severe foliation defects and inferior lobe malformation, characterized by immature Purkinje cells (PCs). Early postnatal Tmem67 mutant cerebellum had disrupted ciliogenesis and reduced responsiveness to Shh signalling. Transcriptome profiling of Tmem67 mutant cerebella identified ectopic increased expression of homeobox-type transcription factors (Hoxa5, Hoxa4, Hoxb5 and Hoxd3), normally required for early rostral hindbrain patterning. HOXB5 protein levels were increased in the inferior lobe, and increased canonical Wnt signalling, following loss of TMEM67, was dependent on HOXB5. HOXB5 occupancy at the ß-catenin promoter was significantly increased by activation of canonical Wnt signalling in Tmem67-/- mutant cerebellar neurones, suggesting that increased canonical Wnt signalling following mutation or loss of TMEM67 was directly dependent on HOXB5. Our results link dysregulated expression of Hox group genes with ciliary Wnt signalling defects in the developing cerebellum, providing new mechanistic insights into ciliopathy cerebellar hypoplasia phenotypes.
Asunto(s)
Cerebelo/embriología , Cilios/metabolismo , Proteínas de Homeodominio/fisiología , Proteínas de la Membrana/fisiología , Transducción de Señal/fisiología , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
OBJECTIVES: The aim of this study was to investigate the distribution of primary cilia on secretory cells in normal fallopian tube (FT) and serous tubal intraepithelial carcinoma (STIC). METHODS: Fallopian tube tissue samples were obtained from 4 females undergoing prophylactic hysterectomies and 6 patients diagnosed with STIC. A mogp-TAg transgenic mouse STIC sample was also compared with a wild-type mouse FT sample. Serous tubal intraepithelial carcinoma was identified by hematoxylin and eosin staining and confirmed by positive Ki-67 and p53 immunohistochemical staining of tissue sections. We assessed the relative distribution of primary cilia on secretory cells and motile cilia on multiple ciliated cells by immunofluorescence and immunohistochemical staining. Ciliary function was assessed by immunofluorescence staining of specific ciliary marker proteins and responsiveness to Sonic Hedgehog signaling. RESULTS: Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of human FT where they may appear to mediate ciliary-mediated Sonic Hedgehog signaling. A statistically significant reduction in the number of primary cilia on secretory cells was observed in human STIC samples compared with normal controls (P < 0.0002, Student t test), supported by similar findings in a mouse STIC sample. Immunohistochemical staining for dynein axonemal heavy chain 5 discriminated multiple motile cilia from primary cilia in human FT. CONCLUSIONS: Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of the human FT but are significantly reduced in both human and mouse STIC samples. Immunohistochemical staining for ciliary proteins may have clinical utility for early detection of STIC.
Asunto(s)
Carcinoma in Situ/patología , Cilios/fisiología , Cistadenocarcinoma Seroso/patología , Neoplasias de las Trompas Uterinas/patología , Trompas Uterinas/citología , Animales , Carcinoma in Situ/metabolismo , Cilios/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Neoplasias de las Trompas Uterinas/metabolismo , Trompas Uterinas/metabolismo , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Ratones , Ratones Transgénicos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Cultivo Primario de Células , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The neuromuscular junction (NMJ) consists of a tripartite synapse with a presynaptic nerve terminal, Schwann cells that ensheathe the terminal bouton, and a highly specialized postsynaptic membrane. Synaptic structural integrity is crucial for efficient signal transmission. Congenital myasthenic syndromes (CMSs) are a heterogeneous group of inherited disorders that result from impaired neuromuscular transmission, caused by mutations in genes encoding proteins that are involved in synaptic transmission and in forming and maintaining the structural integrity of NMJs. To identify further causes of CMSs, we performed whole-exome sequencing (WES) in families without an identified mutation in known CMS-associated genes. In two families affected by a previously undefined CMS, we identified homozygous loss-of-function mutations in COL13A1, which encodes the alpha chain of an atypical non-fibrillar collagen with a single transmembrane domain. COL13A1 localized to the human muscle motor endplate. Using CRISPR-Cas9 genome editing, modeling of the COL13A1 c.1171delG (p.Leu392Sfs(∗)71) frameshift mutation in the C2C12 cell line reduced acetylcholine receptor (AChR) clustering during myotube differentiation. This highlights the crucial role of collagen XIII in the formation and maintenance of the NMJ. Our results therefore delineate a myasthenic disorder that is caused by loss-of-function mutations in COL13A1, encoding a protein involved in organization of the NMJ, and emphasize the importance of appropriate symptomatic treatment for these individuals.
Asunto(s)
Colágeno Tipo XIII/genética , Mutación , Síndromes Miasténicos Congénitos/genética , Mioblastos/metabolismo , Unión Neuromuscular/metabolismo , Adulto , Animales , Línea Celular , Preescolar , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Colágeno Tipo XIII/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Exoma , Femenino , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Masculino , Ratones , Síndromes Miasténicos Congénitos/metabolismo , Síndromes Miasténicos Congénitos/patología , Mioblastos/patología , Unión Neuromuscular/crecimiento & desarrollo , Unión Neuromuscular/patología , Linaje , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Sinapsis/genética , Sinapsis/metabolismo , Sinapsis/patología , Transmisión SinápticaRESUMEN
Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.
Asunto(s)
Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Marcadores Genéticos , Pruebas Genéticas/métodos , Genómica/métodos , Células Fotorreceptoras , Interferencia de ARN , Anomalías Múltiples , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestructura , Enfermedades Cerebelosas/genética , Cerebelo/anomalías , Cilios/metabolismo , Cilios/patología , Trastornos de la Motilidad Ciliar/metabolismo , Trastornos de la Motilidad Ciliar/patología , Proteínas del Citoesqueleto , Bases de Datos Genéticas , Síndrome de Ellis-Van Creveld/genética , Anomalías del Ojo/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Enfermedades Renales Quísticas/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fenotipo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestructura , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Proteínas/genética , Proteínas/metabolismo , Reproducibilidad de los Resultados , Retina/anomalías , Factores Supresores Inmunológicos/genética , Factores Supresores Inmunológicos/metabolismo , Transfección , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Ciliopathies are a group of developmental disorders that manifest with multi-organ anomalies. Mutations in TMEM67 (MKS3) cause a range of human ciliopathies, including Meckel-Gruber and Joubert syndromes. In this study we describe multi-organ developmental abnormalities in the Tmem67(tm1Dgen/H1) knockout mouse that closely resemble those seen in Wnt5a and Ror2 knockout mice. These include pulmonary hypoplasia, ventricular septal defects, shortening of the body longitudinal axis, limb abnormalities, and cochlear hair cell stereociliary bundle orientation and basal body/kinocilium positioning defects. The basal body/kinocilium complex was often uncoupled from the hair bundle, suggesting aberrant basal body migration, although planar cell polarity and apical planar asymmetry in the organ of Corti were normal. TMEM67 (meckelin) is essential for phosphorylation of the non-canonical Wnt receptor ROR2 (receptor-tyrosine-kinase-like orphan receptor 2) upon stimulation with Wnt5a-conditioned medium. ROR2 also colocalises and interacts with TMEM67 at the ciliary transition zone. Additionally, the extracellular N-terminal domain of TMEM67 preferentially binds to Wnt5a in an in vitro binding assay. Cultured lungs of Tmem67 mutant mice failed to respond to stimulation of epithelial branching morphogenesis by Wnt5a. Wnt5a also inhibited both the Shh and canonical Wnt/ß-catenin signalling pathways in wild-type embryonic lung. Pulmonary hypoplasia phenotypes, including loss of correct epithelial branching morphogenesis and cell polarity, were rescued by stimulating the non-canonical Wnt pathway downstream of the Wnt5a-TMEM67-ROR2 axis by activating RhoA. We propose that TMEM67 is a receptor that has a main role in non-canonical Wnt signalling, mediated by Wnt5a and ROR2, and normally represses Shh signalling. Downstream therapeutic targeting of the Wnt5a-TMEM67-ROR2 axis might, therefore, reduce or prevent pulmonary hypoplasia in ciliopathies and other congenital conditions.
Asunto(s)
Tipificación del Cuerpo , Trastornos de la Motilidad Ciliar/metabolismo , Encefalocele/metabolismo , Epitelio/embriología , Proteínas de la Membrana/metabolismo , Morfogénesis , Enfermedades Renales Poliquísticas/metabolismo , Vía de Señalización Wnt , Animales , Animales Recién Nacidos , Diferenciación Celular , Polaridad Celular , Cilios/metabolismo , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/metabolismo , Epitelio/metabolismo , Células HEK293 , Humanos , Pulmón/embriología , Pulmón/metabolismo , Proteínas de la Membrana/deficiencia , Ratones , Mutación/genética , Órgano Espiral/anomalías , Órgano Espiral/embriología , Órgano Espiral/patología , Fenotipo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Retinitis Pigmentosa , Estereocilios/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt-5a , beta Catenina/metabolismoRESUMEN
Vertebrate hedgehog signaling is coordinated by the differential localization of the receptors patched-1 and Smoothened in the primary cilium. Cilia assembly is mediated by intraflagellar transport (IFT), and cilia defects disrupt hedgehog signaling, causing many structural birth defects. We generated Ift25 and Ift27 knockout mice and show that they have structural birth defects indicative of hedgehog signaling dysfunction. Surprisingly, ciliary assembly is not affected, but abnormal hedgehog signaling is observed in conjunction with ciliary accumulation of patched-1 and Smoothened. Similarly, Smoothened accumulates in cilia on cells mutated for BBSome components or the BBS binding protein/regulator Lztfl1. Interestingly, the BBSome and Lztfl1 accumulate to high levels in Ift27 mutant cilia. Because Lztfl1 mutant cells accumulate BBSome but not IFT27, it is likely that Lztfl1 functions downstream of IFT27 to couple the BBSome to the IFT particle for coordinated removal of patched-1 and Smoothened from cilia during hedgehog signaling.
Asunto(s)
Cilios/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rab/metabolismo , Animales , Transporte Biológico , Flagelos/metabolismo , Proteínas Hedgehog/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Superficie Celular/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Mitochondrial Ca(2+) uptake has key roles in cell life and death. Physiological Ca(2+) signaling regulates aerobic metabolism, whereas pathological Ca(2+) overload triggers cell death. Mitochondrial Ca(2+) uptake is mediated by the Ca(2+) uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca(2+)-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca(2+) uptake at low cytosolic Ca(2+) concentrations was increased, and cytosolic Ca(2+) signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca(2+) handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca(2+) signaling, demonstrating the crucial role of mitochondrial Ca(2+) uptake in humans.
Asunto(s)
Señalización del Calcio/genética , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/genética , Discapacidades para el Aprendizaje/genética , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Trastornos del Movimiento/genética , Enfermedades Musculares/genética , Fenotipo , Análisis de Varianza , Secuencia de Bases , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , ADN Complementario/genética , Exoma/genética , Tractos Extrapiramidales/patología , Técnica del Anticuerpo Fluorescente , Técnicas Histológicas , Humanos , Inmunohistoquímica , Potencial de la Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Datos de Secuencia Molecular , Linaje , Polimorfismo de Nucleótido Simple/genética , Músculo Cuádriceps/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Short stature, auditory canal atresia, mandibular hypoplasia, and skeletal abnormalities (SAMS) has been reported previously to be a rare, autosomal-recessive developmental disorder with other, unique rhizomelic skeletal anomalies. These include bilateral humeral hypoplasia, humeroscapular synostosis, pelvic abnormalities, and proximal defects of the femora. To identify the genetic basis of SAMS, we used molecular karyotyping and whole-exome sequencing (WES) to study small, unrelated families. Filtering of variants from the WES data included segregation analysis followed by comparison of in-house exomes. We identified a homozygous 306 kb microdeletion and homozygous predicted null mutations of GSC, encoding Goosecoid homeobox protein, a paired-like homeodomain transcription factor. This confirms that SAMS is a human malformation syndrome resulting from GSC mutations. Previously, Goosecoid has been shown to be a determinant at the Xenopus gastrula organizer region and a segment-polarity determinant in Drosophila. In the present report, we present data on Goosecoid protein localization in staged mouse embryos. These data and the SAMS clinical phenotype both suggest that Goosecoid is a downstream effector of the regulatory networks that define neural-crest cell-fate specification and subsequent mesoderm cell lineages in mammals, particularly during shoulder and hip formation. Our findings confirm that Goosecoid has an essential role in human craniofacial and joint development and suggest that Goosecoid is an essential regulator of mesodermal patterning in mammals and that it has specific functions in neural crest cell derivatives.
Asunto(s)
Anomalías Múltiples/genética , Huesos/anomalías , Enanismo/genética , Conducto Auditivo Externo/anomalías , Proteína Goosecoide/genética , Mandíbula/anomalías , Mutación , Anomalías Múltiples/diagnóstico , Adulto , Animales , Niño , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Homocigoto , Humanos , Masculino , Ratones , Linaje , Fenotipo , Síndrome , Adulto JovenRESUMEN
The ciliopathies are a group of heterogeneous diseases with considerable variations in phenotype for allelic conditions such as Meckel-Gruber syndrome (MKS) and Joubert syndrome (JBTS) even at the inter-individual level within families. In humans, mutations in TMEM67 (also known as MKS3) cause both MKS and JBTS, with TMEM67 encoding the orphan receptor meckelin (TMEM67) that localizes to the ciliary transition zone. We now describe the Tmem67(tm1(Dgen/H)) knockout mouse model that recapitulates the brain phenotypic variability of these human ciliopathies, with categorization of Tmem67 mutant animals into two phenotypic groups. An MKS-like incipient congenic group (F6 to F10) manifested very variable neurological features (including exencephaly, and frontal/occipital encephalocele) that were associated with the loss of primary cilia, diminished Shh signalling and dorsalization of the caudal neural tube. The 'MKS-like' group also had high de-regulated canonical Wnt/ß-catenin signalling associated with hyper-activated Dishevelled-1 (Dvl-1) localized to the basal body. Conversely, a second fully congenic group (F > 10) had less variable features pathognomonic for JBTS (including cerebellar hypoplasia), and retention of abnormal bulbous cilia associated with mild neural tube ventralization. The 'JBTS-like' group had de-regulated low levels of canonical Wnt signalling associated with the loss of Dvl-1 localization to the basal body. Our results suggest that modifier alleles partially determine the variation between MKS and JBTS, implicating the interaction between Dvl-1 and meckelin, or other components of the ciliary transition zone. The Tmem67(tm1(Dgen/H)) line is unique in modelling the variable expressivity of phenotypes in these two ciliopathies.
Asunto(s)
Enfermedades Cerebelosas/metabolismo , Trastornos de la Motilidad Ciliar/metabolismo , Encefalocele/metabolismo , Anomalías del Ojo/metabolismo , Proteínas Hedgehog/metabolismo , Enfermedades Renales Quísticas/metabolismo , Proteínas de la Membrana/genética , Enfermedades Renales Poliquísticas/metabolismo , Vía de Señalización Wnt , Anomalías Múltiples , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Tipificación del Cuerpo/genética , Enfermedades Cerebelosas/genética , Enfermedades Cerebelosas/patología , Cerebelo/anomalías , Cilios/patología , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/patología , Modelos Animales de Enfermedad , Proteínas Dishevelled , Encefalocele/genética , Encefalocele/patología , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , Humanos , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/patología , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Proteínas de la Membrana/deficiencia , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/metabolismo , Defectos del Tubo Neural/patología , Fenotipo , Fosfoproteínas/metabolismo , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Transporte de Proteínas , Retina/anomalías , Retina/metabolismo , Retina/patología , Retinitis PigmentosaRESUMEN
Infantile myopathies with diaphragmatic paralysis are genetically heterogeneous, and clinical symptoms do not assist in differentiating between them. We used phased haplotype analysis with subsequent targeted exome sequencing to identify MEGF10 mutations in a previously unidentified type of infantile myopathy with diaphragmatic weakness, areflexia, respiratory distress and dysphagia. MEGF10 is highly expressed in activated satellite cells and regulates their proliferation as well as their differentiation and fusion into multinucleated myofibers, which are greatly reduced in muscle from individuals with early onset myopathy, areflexia, respiratory distress and dysphagia.