RESUMEN
Biallelic loss-of-function mutations in the RNA-binding protein EIF4A3 cause Richieri-Costa-Pereira syndrome (RCPS), an autosomal recessive condition mainly characterized by craniofacial and limb malformations. However, the pathogenic cellular mechanisms responsible for this syndrome are entirely unknown. Here, we used two complementary approaches, patient-derived induced pluripotent stem cells (iPSCs) and conditional Eif4a3 mouse models, to demonstrate that defective neural crest cell (NCC) development explains RCPS craniofacial abnormalities. RCPS iNCCs have decreased migratory capacity, a distinct phenotype relative to other craniofacial disorders. Eif4a3 haploinsufficient embryos presented altered mandibular process fusion and micrognathia, thus recapitulating the most penetrant phenotypes of the syndrome. These defects were evident in either ubiquitous or NCC-specific Eif4a3 haploinsufficient animals, demonstrating an autonomous requirement of Eif4a3 in NCCs. Notably, RCPS NCC-derived mesenchymal stem-like cells (nMSCs) showed premature bone differentiation, a phenotype paralleled by premature clavicle ossification in Eif4a3 haploinsufficient embryos. Likewise, nMSCs presented compromised in vitro chondrogenesis, and Meckel's cartilage was underdeveloped in vivo. These findings indicate novel and essential requirements of EIF4A3 for NCC migration and osteochondrogenic differentiation during craniofacial development. Altogether, complementary use of iPSCs and mouse models pinpoint unique cellular mechanisms by which EIF4A3 mutation causes RCPS, and provide a paradigm to study craniofacial disorders.
Asunto(s)
Pie Equinovaro/genética , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Deformidades Congénitas de la Mano/genética , Síndrome de Pierre Robin/genética , Animales , Huesos/metabolismo , Región Branquial/metabolismo , Diferenciación Celular/genética , Movimiento Celular , Condrogénesis/genética , Pie Equinovaro/metabolismo , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/metabolismo , Modelos Animales de Enfermedad , Deformidades Congénitas de la Mano/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Cresta Neural/crecimiento & desarrollo , Cresta Neural/metabolismo , Osteogénesis/genética , Síndrome de Pierre Robin/metabolismoRESUMEN
Treacher Collins Syndrome (TCS) is a rare congenital disease (1:50 000 live births) characterized by craniofacial defects, including hypoplasia of facial bones, cleft palate and palpebral fissures. Over 90% of the cases are due to mutations in the TCOF1 gene, which codifies the nucleolar protein Treacle. Here we report a novel TCS-like zebrafish model displaying features that fully recapitulate the spectrum of craniofacial abnormalities observed in patients. As it was reported for a Tcof1+/- mouse model, Treacle depletion in zebrafish caused reduced rRNA transcription, stabilization of Tp53 and increased cell death in the cephalic region. An increase of ROS along with the overexpression of redox-responsive genes was detected; furthermore, treatment with antioxidants ameliorated the phenotypic defects of craniofacial anomalies in TCS-like larvae. On the other hand, Treacle depletion led to a lowering in the abundance of Cnbp, a protein required for proper craniofacial development. Tcof1 knockdown in transgenic zebrafish overexpressing cnbp resulted in barely affected craniofacial cartilage development, reinforcing the notion that Cnbp has a role in the pathogenesis of TCS. The cnbp overexpression rescued the TCS phenotype in a dose-dependent manner by a ROS-cytoprotective action that prevented the redox-responsive genes' upregulation but did not normalize the synthesis of rRNAs. Finally, a positive correlation between the expression of CNBP and TCOF1 in mesenchymal cells from both control and TCS subjects was found. Based on this, we suggest CNBP as an additional target for new alternative therapeutic treatments to reduce craniofacial defects not only in TCS but also in other neurocristopathies.
Asunto(s)
Anomalías Craneofaciales/complicaciones , Anomalías Craneofaciales/genética , Disostosis Mandibulofacial/complicaciones , Disostosis Mandibulofacial/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genética , Proteínas de Pez Cebra/metabolismo , Animales , Anomalías Craneofaciales/patología , Embrión no Mamífero/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Disostosis Mandibulofacial/patología , Mesodermo/metabolismo , Modelos Biológicos , Oxidación-Reducción , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Gain-of-function mutations in FGFR2 cause Apert syndrome (AS), a disease characterized by craniosynostosis and limb bone defects both due to abnormalities in bone differentiation and remodeling. Although the periosteum is an important cell source for bone remodeling, its role in craniosynostosis remains poorly characterized. We hypothesized that periosteal mesenchymal stem cells (MSCs) and fibroblasts from AS patients have abnormal cell phenotypes that contribute to the recurrent fusion of the coronal sutures. MSCs and fibroblasts were obtained from the periostea of 3 AS patients (S252W) and 3 control individuals (WT). We evaluated the proliferation, migration, and osteogenic differentiation of these cells. Interestingly, S252W mutation had opposite effects on different cell types: S252W MSCs proliferated less than WT MSCs, while S252W fibroblasts proliferated more than WT fibroblasts. Under restrictive media conditions, only S252W fibroblasts showed enhanced migration. The presence of S252W mutation increased in vitro and in vivo osteogenic differentiation in both studied cell types, though the difference compared to WT cells was more pronounced in S252W fibroblasts. This osteogenic differentiation was reversed through inhibition of JNK. We demonstrated that S252W fibroblasts can induce osteogenic differentiation in periosteal MSCs but not in MSCs from another tissue. MSCs and fibroblasts responded differently to the pathogenic effects of the FGFR2(S252W) mutation. We propose that cells from the periosteum have a more important role in the premature fusion of cranial sutures than previously thought and that molecules in JNK pathway are strong candidates for the treatment of AS patients.