RESUMEN
Piezo1 and 2 are evolutionarily conserved mechanosensory cation channels known to function on the cell surface by responding to external pressure and transducing a mechanically activated Ca2+ current. Here we show that both Piezo1 and 2 also exhibit concentrated intracellular localization at centrosomes. Both Piezo1 and 2 loss-of-function and Piezo1 activation by the small molecule Yoda1 result in supernumerary centrosomes, premature centriole disengagement, multi-polar spindles, and mitotic delay. By using a GFP, Calmodulin and M13 Protein fusion (GCaMP) Ca2+-sensitive reporter, we show that perturbations in Piezo modulate Ca2+ flux at centrosomes. Moreover, the inhibition of Polo-like-kinase 1 eliminates Yoda1-induced centriole disengagement. Because previous studies have implicated force generation by microtubules as essential for maintaining centrosomal integrity, we propose that mechanotransduction by Piezo maintains pericentrosomal Ca2+ within a defined range, possibly through sensing cell intrinsic forces from microtubules.
Asunto(s)
Centrosoma , Mecanotransducción Celular , Centrosoma/metabolismo , Centriolos , MicrotúbulosRESUMEN
Primary glomerulocystic kidney disease is a special form of renal cystic disorder characterized by Bowman's space dilatation in the absence of tubular cysts. ZEB2 is a SMAD-interacting transcription factor involved in Mowat-Wilson syndrome, a congenital disorder with an increased risk for kidney anomalies. Here we show that deletion of Zeb2 in mesenchyme-derived nephrons with either Pax2-cre or Six2-cre causes primary glomerulocystic kidney disease without tubular cysts in mice. Glomerulotubular junction analysis revealed many atubular glomeruli in the kidneys of Zeb2 knockout mice, which explains the presence of glomerular cysts in the absence of tubular dilatation. Gene expression analysis showed decreased expression of early proximal tubular markers in the kidneys of Zeb2 knockout mice preceding glomerular cyst formation, suggesting that defects in proximal tubule development during early nephrogenesis contribute to the formation of congenital atubular glomeruli. At the molecular level, Zeb2 deletion caused aberrant expression of Pkd1, Hnf1ß, and Glis3, three genes causing glomerular cysts. Thus, Zeb2 regulates the morphogenesis of mesenchyme-derived nephrons and is required for proximal tubule development and glomerulotubular junction formation. Our findings also suggest that ZEB2 might be a novel disease gene in patients with primary glomerular cystic disease.
Asunto(s)
Enfermedades del Sistema Nervioso Central/genética , Esmalte Dental/anomalías , Diabetes Mellitus Tipo 2/genética , Proteínas de Homeodominio/fisiología , Enfermedades Renales Quísticas/genética , Riñón/embriología , Proteínas Represoras/fisiología , Animales , Proteínas de Unión al ADN , Factor Nuclear 1-beta del Hepatocito/metabolismo , Riñón/metabolismo , Ratones Noqueados , Proteínas Represoras/metabolismo , Canales Catiónicos TRPP/metabolismo , Transactivadores/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
CAPZB is an actin-capping protein that caps the growing end of F-actin and modulates the cytoskeleton and tethers actin filaments to the Z-line of the sarcomere in muscles. Whole-genome sequencing was performed on a subject with micrognathia, cleft palate and hypotonia that harbored a de novo, balanced chromosomal translocation that disrupts the CAPZB gene. The function of capzb was analyzed in the zebrafish model. capzb(-/-) mutants exhibit both craniofacial and muscle defects that recapitulate the phenotypes observed in the human subject. Loss of capzb affects cell morphology, differentiation and neural crest migration. Differentiation of both myogenic stem cells and neural crest cells requires capzb. During palate morphogenesis, defective cranial neural crest cell migration in capzb(-/-) mutants results in loss of the median cell population, creating a cleft phenotype. capzb is also required for trunk neural crest migration, as evident from melanophores disorganization in capzb(-/-) mutants. In addition, capzb over-expression results in embryonic lethality. Therefore, proper capzb dosage is important during embryogenesis, and regulates both cell behavior and tissue morphogenesis.
Asunto(s)
Proteína CapZ/genética , Diferenciación Celular , Cabeza/embriología , Morfogénesis , Cresta Neural/embriología , Animales , Fisura del Paladar/genética , Fisura del Paladar/metabolismo , Modelos Animales de Enfermedad , Femenino , Cabeza/fisiología , Humanos , Lactante , Micrognatismo/genética , Micrognatismo/metabolismo , Hipotonía Muscular/genética , Hipotonía Muscular/metabolismo , Mutación , Cresta Neural/metabolismo , Cresta Neural/fisiología , Análisis de Secuencia de ADN , Síndrome , Pez Cebra/embriología , Pez Cebra/metabolismo , Pez Cebra/fisiologíaRESUMEN
The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1(glcr11), which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1(null) and Crim1(cko), we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active ß1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Cristalino/citología , Animales , Receptores de Proteínas Morfogenéticas Óseas/genética , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Cristalino/metabolismo , Ratones , Ratones Endogámicos C57BL , Organogénesis/genética , Organogénesis/fisiología , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
Cardiac left ventricular outflow tract (LVOT) defects represent a common but heterogeneous subset of congenital heart disease for which gene identification has been difficult. We describe a 46,XY,t(1;5)(p36.11;q31.2)dn translocation carrier with pervasive developmental delay who also exhibited LVOT defects, including bicuspid aortic valve (BAV), coarctation of the aorta (CoA) and patent ductus arteriosus (PDA). The 1p breakpoint disrupts the 5' UTR of AHDC1, which encodes AT-hook DNA-binding motif containing-1 protein, and AHDC1-truncating mutations have recently been described in a syndrome that includes developmental delay, but not congenital heart disease [Xia, F., Bainbridge, M.N., Tan, T.Y., Wangler, M.F., Scheuerle, A.E., Zackai, E.H., Harr, M.H., Sutton, V.R., Nalam, R.L., Zhu, W. et al. (2014) De Novo truncating mutations in AHDC1 in individuals with syndromic expressive language delay, hypotonia, and sleep apnea. Am. J. Hum. Genet., 94, 784-789]. On the other hand, the 5q translocation breakpoint disrupts the 3' UTR of MATR3, which encodes the nuclear matrix protein Matrin 3, and mouse Matr3 is strongly expressed in neural crest, developing heart and great vessels, whereas Ahdc1 is not. To further establish MATR3 3' UTR disruption as the cause of the proband's LVOT defects, we prepared a mouse Matr3(Gt-ex13) gene trap allele that disrupted the 3' portion of the gene. Matr3(Gt-ex13) homozygotes are early embryo lethal, but Matr3(Gt-ex13) heterozygotes exhibit incompletely penetrant BAV, CoA and PDA phenotypes similar to those in the human proband, as well as ventricular septal defect (VSD) and double-outlet right ventricle (DORV). Both the human MATR3 translocation breakpoint and the mouse Matr3(Gt-ex13) gene trap insertion disturb the polyadenylation of MATR3 transcripts and alter Matrin 3 protein expression, quantitatively or qualitatively. Thus, subtle perturbations in Matrin 3 expression appear to cause similar LVOT defects in human and mouse.
Asunto(s)
Coartación Aórtica/genética , Válvula Aórtica/anomalías , Conducto Arterioso Permeable/genética , Enfermedades de las Válvulas Cardíacas/genética , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas de Unión al ARN/genética , Adolescente , Animales , Coartación Aórtica/metabolismo , Válvula Aórtica/metabolismo , Enfermedad de la Válvula Aórtica Bicúspide , Preescolar , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Conducto Arterioso Permeable/metabolismo , Femenino , Silenciador del Gen , Enfermedades de las Válvulas Cardíacas/metabolismo , Ventrículos Cardíacos/anomalías , Ventrículos Cardíacos/metabolismo , Humanos , Recién Nacido , Masculino , Ratones , Mutagénesis Insercional , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Translocación GenéticaRESUMEN
Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and ß-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.
Asunto(s)
Células Madre Embrionarias/fisiología , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Cristalino/fisiología , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Animales , Western Blotting , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Madre Embrionarias/citología , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Humanos , Cristalino/citología , Ratones , Proteínas del Tejido Nervioso/genética , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Homeobox SIX3RESUMEN
We describe a male patient (patient DGAP113) with a balanced translocation, 46,XY,t(1;3)(q31.3;q13.13), severe bilateral congenital cataracts, CNS abnormalities and mild developmental delay. Fluorescence in situ hybridization (FISH) and suppression PCR demonstrated that the chromosome 3 breakpoint lies ~515 kb upstream of the PVRL3 gene, while the chromosome 1 breakpoint lies ~50 kb upstream of the NEK7 gene. Despite the fact that NEK7 is closer to a translocation breakpoint than PVRL3, NEK7 transcript levels are unaltered in patient DGAP113 lymphoblastoid cells and Nek7-deficient mice exhibit no detectable ocular phenotype. In contrast, the expression of PVRL3, which encodes the cell adhesion protein Nectin 3, is significantly reduced in patient DGAP113 lymphoblastoid cells, likely due to a position effect caused by the chromosomal translocation. Nectin 3 is expressed in the mouse embryonic ciliary body and lens. Moreover, Pvrl3 knockout mice as well as a spontaneous mouse mutant ari (anterior retinal inversion), that maps to the Pvrl3 locus, exhibit lens and other ocular defects involving the ciliary body. Collectively, these data identify PVRL3 as a critical gene involved in a Nectin-mediated cell-cell adhesion mechanism in human ocular development.
Asunto(s)
Catarata/congénito , Catarata/genética , Moléculas de Adhesión Celular/genética , Animales , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Rotura Cromosómica , Humanos , Linfocitos , Masculino , Ratones , Mutación , Quinasas Relacionadas con NIMA , Nectinas , Proteínas Serina-Treonina Quinasas/metabolismo , Translocación GenéticaRESUMEN
Complex central nervous system (CNS) malformations frequently coexist with other developmental abnormalities, but whether the associated defects share a common genetic basis is often unclear. We describe five individuals who share phenotypically related CNS malformations and in some cases urinary tract defects, and also haploinsufficiency for the NFIA transcription factor gene due to chromosomal translocation or deletion. Two individuals have balanced translocations that disrupt NFIA. A third individual and two half-siblings in an unrelated family have interstitial microdeletions that include NFIA. All five individuals exhibit similar CNS malformations consisting of a thin, hypoplastic, or absent corpus callosum, and hydrocephalus or ventriculomegaly. The majority of these individuals also exhibit Chiari type I malformation, tethered spinal cord, and urinary tract defects that include vesicoureteral reflux. Other genes are also broken or deleted in all five individuals, and may contribute to the phenotype. However, the only common genetic defect is NFIA haploinsufficiency. In addition, previous analyses of Nfia(-/-) knockout mice indicate that Nfia deficiency also results in hydrocephalus and agenesis of the corpus callosum. Further investigation of the mouse Nfia(+/-) and Nfia(-/-) phenotypes now reveals that, at reduced penetrance, Nfia is also required in a dosage-sensitive manner for ureteral and renal development. Nfia is expressed in the developing ureter and metanephric mesenchyme, and Nfia(+/-) and Nfia(-/-) mice exhibit abnormalities of the ureteropelvic and ureterovesical junctions, as well as bifid and megaureter. Collectively, the mouse Nfia mutant phenotype and the common features among these five human cases indicate that NFIA haploinsufficiency contributes to a novel human CNS malformation syndrome that can also include ureteral and renal defects.
Asunto(s)
Anomalías Múltiples/genética , Predisposición Genética a la Enfermedad , Haploidia , Factores de Transcripción NFI/genética , Malformaciones del Sistema Nervioso/genética , Anomalías Urogenitales/genética , Animales , Niño , Preescolar , Cromosomas Humanos Par 1/genética , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Reordenamiento Génico , Humanos , Lactante , Riñón/anomalías , Riñón/embriología , Riñón/metabolismo , Masculino , Ratones , Mutación/genética , Factores de Transcripción NFI/metabolismo , Fenotipo , Médula Espinal/metabolismo , Síndrome , Uréter/anomalías , Uréter/embriología , Uréter/metabolismo , Uréter/patologíaRESUMEN
Congenital anomalies of the kidney and urinary tract (CAKUT) include vesicoureteral reflux (VUR). VUR is a complex, genetically heterogeneous developmental disorder characterized by the retrograde flow of urine from the bladder into the ureter and is associated with reflux nephropathy, the cause of 15% of end-stage renal disease in children and young adults. We investigated a man with a de novo translocation, 46,X,t(Y;3)(p11;p12)dn, who exhibits multiple congenital abnormalities, including severe bilateral VUR with ureterovesical junction defects. This translocation disrupts ROBO2, which encodes a transmembrane receptor for SLIT ligand, and produces dominant-negative ROBO2 proteins that abrogate SLIT-ROBO signaling in vitro. In addition, we identified two novel ROBO2 intracellular missense variants that segregate with CAKUT and VUR in two unrelated families. Adult heterozygous and mosaic mutant mice with reduced Robo2 gene dosage also exhibit striking CAKUT-VUR phenotypes. Collectively, these results implicate the SLIT-ROBO signaling pathway in the pathogenesis of a subset of human VUR.