RESUMEN
BACKGROUND: Orofacial clefts are one of the most common congenital malformations of the fetal face and ultrasound is mainly responsible for its diagnosis. It is difficult to view the fetal palate, so there is currently no unified standard for fetal palate screening, and the diagnosis of cleft palate is not included in the relevant prenatal ultrasound screening guidelines. Many prenatal diagnoses for cleft palate are missed due to the lack of effective screening methods. Therefore, it is imperative to increase the display rate of the fetal palate, which would improve the detection rate and diagnostic accuracy for cleft palate. We aim to introduce a fetal palate screening software based on the "sequential sector scan though the oral fissure", an effective method for fetal palate screening which was verified by our follow up results and three-dimensional ultrasound and to evaluate its feasibility and clinical practicability. METHODS: A software was designed and programmed based on "sequential sector scan through the oral fissure" and three-dimensional ultrasound. The three-dimensional ultrasound volume data of the fetal face were imported into the software. Then, the median sagittal plane was taken as the reference interface, the anterior upper margin of the mandibular alveolar bone was selected as the fulcrum, the interval angles, and the number of layers of the sector scan were set, after which the automatic scan was performed. Thus, the sector scan sequential planes of the mandibular alveolar bone, pharynx, soft palate, hard palate, and maxillary alveolar bone were obtained in sequence to display and evaluate the palate. In addition, the feasibility and accuracy of the software in fetal palate displaying and screening was evaluated by actual clinical cases. RESULTS: Full views of the normal fetal palates and the defective parts of the cleft palates were displayed, and relatively clear sequential tomographic images and continuous dynamic videos were formed after the three-dimensional volume data of 10 normal fetal palates and 10 cleft palates were imported into the software. CONCLUSIONS: The software can display fetal palates more directly which might allow for a new method of fetal palate screening and cleft palate diagnosis.
Asunto(s)
Fisura del Paladar , Imagenología Tridimensional , Programas Informáticos , Ultrasonografía Prenatal , Humanos , Ultrasonografía Prenatal/métodos , Fisura del Paladar/diagnóstico por imagen , Fisura del Paladar/embriología , Imagenología Tridimensional/métodos , Embarazo , Femenino , Hueso Paladar/diagnóstico por imagen , Hueso Paladar/embriología , Adulto , Estudios de FactibilidadRESUMEN
Orofacial clefts (OFCs) are among the most common human congenital birth defects. Previous multiethnic studies have identified dozens of associated loci for both cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP). Although several nearby genes have been highlighted, the "casual" variants are largely unknown. Here, we developed DeepFace, a convolutional neural network model, to assess the functional impact of variants by SNP activity difference (SAD) scores. The DeepFace model is trained with 204 epigenomic assays from crucial human embryonic craniofacial developmental stages of post-conception week (pcw) 4 to pcw 10. The Pearson correlation coefficient between the predicted and actual values for 12 epigenetic features achieved a median range of 0.50-0.83. Specifically, our model revealed that SNPs significantly associated with OFCs tended to exhibit higher SAD scores across various variant categories compared to less related groups, indicating a context-specific impact of OFC-related SNPs. Notably, we identified six SNPs with a significant linear relationship to SAD scores throughout developmental progression, suggesting that these SNPs could play a temporal regulatory role. Furthermore, our cell-type specificity analysis pinpointed the trophoblast cell as having the highest enrichment of risk signals associated with OFCs. Overall, DeepFace can harness distal regulatory signals from extensive epigenomic assays, offering new perspectives for prioritizing OFC variants using contextualized functional genomic features. We expect DeepFace to be instrumental in accessing and predicting the regulatory roles of variants associated with OFCs, and the model can be extended to study other complex diseases or traits.
Asunto(s)
Labio Leporino , Fisura del Paladar , Aprendizaje Profundo , Polimorfismo de Nucleótido Simple , Humanos , Fisura del Paladar/genética , Fisura del Paladar/embriología , Labio Leporino/genética , Labio Leporino/embriología , Redes Neurales de la Computación , Epigenómica/métodos , Desarrollo Embrionario/genéticaRESUMEN
CDK13-related disorder, also known as congenital heart defects, dysmorphic facial features and intellectual developmental disorder (CHDFIDD) is associated with mutations in the CDK13 gene encoding transcription-regulating cyclin-dependent kinase 13 (CDK13). Here, we focused on the development of craniofacial structures and analyzed early embryonic stages in CHDFIDD mouse models, with one model comprising a hypomorphic mutation in Cdk13 and exhibiting cleft lip/palate, and another model comprising knockout of Cdk13, featuring a stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically expressed at high levels in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13 deficiency leads to development of hypoplastic branches of the trigeminal nerve including the maxillary branch. Additionally, we detected significant changes in the expression levels of genes involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes in the expression pattern of other key face-specific genes (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in the regulation of craniofacial morphogenesis.
Asunto(s)
Modelos Animales de Enfermedad , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Neurogénesis , Animales , Neurogénesis/genética , Desarrollo Embrionario/genética , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , Cráneo/embriología , Cráneo/patología , Ratones , Fisura del Paladar/genética , Fisura del Paladar/patología , Fisura del Paladar/embriología , Labio Leporino/genética , Labio Leporino/patología , Labio Leporino/embriología , Nervio Trigémino/embriología , Embrión de Mamíferos/metabolismo , Cara/embriología , Cara/anomalías , Fenotipo , Discapacidad Intelectual/genética , Mutación/genética , Proteína DoblecortinaRESUMEN
La formación del paladar ocurre entre la quinta y undécima semana de vida intrauterina producto de la unión del paladar primario y secundario. Por otra parte, la formación del labio superior ocurre entre la quinta y sexta semana del desarrollo, y se configura en su parte media por la fusión de los procesos nasales mediales y lateralmente, a expensas de los procesos maxilares. La prevalencia de las fisuras labiales y/o fisura palatina varía según las distintas etnias, con cifras entre 0,7 hasta 1,1 casos por 1000 nacidos vivos. El objetivo de este trabajo fue realizar una revisión bibliográfica sobre aspectos epidemiológicos, mecanismos genéticos moleculares y ambientales que influyen en la ocurrencia de la fisura labial, fisura palatina y fisura labio palatina. La búsqueda bibliográfica se realizó en las bases de datos PUBMED, SCOPUS, SPRINGER, SCIENCEDIRECT utilizando los términos en inglés "cleft lip and palate", "cleft lip", "cleft palate" y "embriology". Entre los criterios de inclusión se consideraron estudios realizados en humanos y animales, publicados entre los años 2015 y 2021. La búsqueda arrojó un total de 407 trabajos, de los cuales tras un filtro por título y resumen quedaron un total de 38 artículos, en los cuales se realizó un análisis de texto completo para finalmente seleccionar 26 artículos que abarcan temas genéticos-moleculares, ambientales, epidemiológicos y sindrómicos. Además se incorporaron por búsqueda manual, 6 documentos asociados a libros de texto, y artículos científicos, sin considerar el criterio inclusión de tiempo. Dentro de esta revisión se describe la fuerte asociación entre las fisuras orales y las mutaciones de genes Msx1, sonic hedgehog, proteínas morfogenéticas óseas y factor de crecimiento fibroblástico durante la migración de las células de la cresta neural y la modelación y formación del paladar. La ausencia de ácido fólico durante el desarrollo del paladar y la presencia de hipoxia por exposición a humo, son los factores ambientales observados con mayor frecuencia en malformaciones orofaciales.
SUMMARY: Palate formation occurs between the fifth and eleventh week of intrauterine life as a result of the union of the primary and secondary palate. On the other hand, the formation of the upper lip occurs between the fifth and sixth week of development, and is configured in its middle part by the fusion of the medial and lateral nasal processes, at the expense of the maxillary processes. The prevalence of cleft lips and / or cleft palate varies according to the different ethnic groups, with figures ranging from 0.7 to 1.1 cases per 1000 live births. The aim of this work was to carry out a literature review on epidemiological aspects, molecular and environmental genetic mechanisms that influence the occurrence of cleft lip, cleft palate and its embriology. The literature search was carried out in the databases PUBMED, SCOPUS, SPRINGER, SCIENCEDIRECT using the English terms "cleft lip and palate", "cleft lip", "cleft palate" and "embryology". Inclusion criteria included studies carried out in humans and animals, published between 2015 and 2021. The search yielded a total of 407 works, of which after a filter by title and abstract, a total of 38 articles remained, in which a text analysis was carried out complete to finally select 26 articles that cover genetic-molecular, environmental, epidemiological and syndromic topics. In addition, 6 documents associated with textbooks and scientific articles were incorporated by manual search, without considering the inclusion criterion of time. This review describes the strong association between oral fissures and mutations of genes Msx1, sonic hedgehog, bone morphogenetic proteins and fibroblast growth factor during migration of neural crest cells and palate shaping and formation. Lack of folic acid during palae development dar and the presence of hypoxia due to exposure to smoke, are the environmental factors most frequently observed in orofacial malformations.
Asunto(s)
Humanos , Labio Leporino/embriología , Fisura del Paladar/embriología , Labio Leporino/genética , Labio Leporino/epidemiología , Fisura del Paladar/genética , Fisura del Paladar/epidemiologíaRESUMEN
Deletions of chromosome 1p36 are the most common telomeric deletions in humans and are associated with an increased risk of orofacial clefting. Deletion/phenotype mapping, combined with data from human and mouse studies, suggests the existence of multiple 1p36 genes associated with orofacial clefting including SKI, PRDM16, PAX7 and GRHL3. The arginine-glutamic acid dipeptide (RE) repeats gene (RERE) is located in the proximal critical region for 1p36 deletion syndrome and encodes a nuclear receptor co-regulator. Pathogenic RERE variants have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye or heart (NEDBEH). Cleft lip has previously been described in one individual with NEDBEH. Here we report the first individual with NEDBEH to have a cleft palate. We confirm that RERE is broadly expressed in the palate during mouse embryonic development, and we demonstrate that the majority of RERE-deficient mouse embryos on C57BL/6 background have cleft palate. We go on to show that ablation of Rere in cranial neural crest (CNC) cells, mediated by a Wnt1-Cre, leads to delayed elevation of the palatal shelves and cleft palate and that proliferation of mesenchymal cells in the palatal shelves is significantly reduced in Rereflox/flox; Wnt1-Cre embryos. We conclude that loss of RERE function contributes to the development of orofacial clefts in individuals with proximal 1p36 deletions and NEDBEH and that RERE expression in CNC cells and their derivatives is required for normal palatal development.
Asunto(s)
Trastornos de los Cromosomas/genética , Labio Leporino/genética , Fisura del Paladar/genética , Modelos Animales de Enfermedad , Desarrollo Embrionario/genética , Proteínas del Tejido Nervioso/genética , Proteínas Represoras/genética , Animales , Proliferación Celular/genética , Deleción Cromosómica , Trastornos de los Cromosomas/metabolismo , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 1/metabolismo , Labio Leporino/embriología , Labio Leporino/metabolismo , Fisura del Paladar/embriología , Fisura del Paladar/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/deficiencia , Cresta Neural/embriología , Cresta Neural/metabolismo , Fenotipo , Proteínas Represoras/deficiencia , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
Cleft lip and/or palate (CL/P) are common anomalies occurring in 1/800 live-births. Pathogenic SPECC1L variants have been identified in patients with CL/P, which signifies a primary role for SPECC1L in craniofacial development. Specc1l mutant mouse embryos exhibit delayed palatal shelf elevation accompanied by epithelial defects. We now posit that the process of palate elevation is itself abnormal in Specc1l mutants, due to defective remodeling of palatal mesenchyme. To characterize the underlying cellular defect, we studied the movement of primary mouse embryonic palatal mesenchyme (MEPM) cells using live-imaging of wound-repair assays. SPECC1L-deficient MEPM cells exhibited delayed wound-repair, however, reduced cell speed only partially accounted for this delay. Interestingly, mutant MEPM cells were also defective in coordinated cell movement. Therefore, we used open-field 2D cultures of wildtype MEPM cells to show that they indeed formed cell streams at high density, which is an important attribute of collective movement. Furthermore, activation of the PI3K-AKT pathway rescued both cell speed and guidance defects in Specc1l mutant MEPM cells. Thus, we show that live-imaging of primary MEPM cells can be used to assess mesenchymal remodeling defects during palatal shelf elevation, and identify a novel role for SPECC1L in collective movement through modulation of PI3K-AKT signaling.
Asunto(s)
Labio Leporino/embriología , Fisura del Paladar/embriología , Embrión de Mamíferos/embriología , Regulación del Desarrollo de la Expresión Génica , Hueso Paladar/embriología , Fosfoproteínas/deficiencia , Animales , Labio Leporino/genética , Fisura del Paladar/genética , Ratones , Ratones Noqueados , Fosfoproteínas/metabolismoAsunto(s)
Enfermedades Cerebelosas/diagnóstico por imagen , Trastornos de los Cromosomas/diagnóstico por imagen , Fisura del Paladar/diagnóstico por imagen , Pérdida Auditiva Central/diagnóstico por imagen , Cardiopatías Congénitas/diagnóstico por imagen , Discapacidad Intelectual/diagnóstico por imagen , Imagen por Resonancia Magnética , Hipotonía Muscular/diagnóstico por imagen , Ultrasonografía Prenatal , Adulto , Enfermedades Cerebelosas/embriología , Trastornos de los Cromosomas/embriología , Fisura del Paladar/embriología , Femenino , Pérdida Auditiva Central/embriología , Cardiopatías Congénitas/embriología , Humanos , Discapacidad Intelectual/embriología , Ilustración Médica , Hipotonía Muscular/embriología , Fenotipo , Embarazo , SíndromeAsunto(s)
Encéfalo/anomalías , Labio Leporino/diagnóstico por imagen , Fisura del Paladar/diagnóstico por imagen , Medida de Translucencia Nucal , Primer Trimestre del Embarazo , Adulto , Encéfalo/diagnóstico por imagen , Encéfalo/embriología , Labio Leporino/embriología , Fisura del Paladar/embriología , Toma de Decisiones , Diagnóstico Precoz , Femenino , Feto/anomalías , Feto/diagnóstico por imagen , Humanos , Valor Predictivo de las Pruebas , Embarazo , Estudios ProspectivosRESUMEN
As the major receptor mediated BMP signaling in craniofacial development, Bmpr1a expression was detected in the anterior palatal shelves from E13.5 and the posterior palatal shelves from E14.5. However, inactivating BMP receptor in the mesenchyme only leads to anterior cleft palate or submucous cleft palate. The role of BMP signaling in posterior palatal mesenchyme and palatal osteogenesis is still unknown. In this study, a secreted BMP antagonist, Noggin was over-expressed by Osr2-creKI to suppress BMP signaling intensively in mouse palatal mesenchyme, which made the newborn mouse displaying complete cleft palate, a phenotype much severer than the anterior or submucous cleft palate. Immunohistochemical analysis indicated that in the anterior and posterior palatal mesenchyme, the canonical BMP-Smad4 signaling was dramatically down-regulated, while the non-canonical BMP signaling pathways were altered little. Although cell proliferation was reduced only in the anterior palatal mesenchyme, the osteogenic condensation and Osterix distribution were remarkably repressed in the posterior palatal mesenchyme by Noggin over-expression. These findings suggested that BMP-Smad4 signaling was essential for the cell proliferation in the anterior palatal mesenchyme, and for the osteogenesis in the posterior palatal mesenchyme. Interestingly, the constitutive activation of Bmpr1a in palatal mesenchyme also caused the complete cleft palate, in which the enhanced BMP-Smad4 signaling resulted in the premature osteogenic differentiation in palatal mesenchyme. Moreover, neither the Noggin over-expression nor Bmpr1a activation disrupted the elevation of palatal shelves. Our study not only suggested that BMP signaling played the differential roles in the anterior and posterior palatal mesenchyme, but also indicated that BMP-Smad4 signaling was required to be finely tuned for the osteogenesis of palatal mesenchyme.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Fisura del Paladar/metabolismo , Mesodermo/patología , Osteogénesis , Hueso Paladar/patología , Transducción de Señal , Proteína Smad4/metabolismo , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Huesos/patología , Bromodesoxiuridina/metabolismo , Proteínas Portadoras/metabolismo , Diferenciación Celular , Proliferación Celular , Fisura del Paladar/embriología , Fisura del Paladar/patología , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Integrasas/metabolismo , Masculino , Mesodermo/embriología , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Tamaño de los Órganos , Hueso Paladar/embriología , Factor de Transcripción Sp7/metabolismoRESUMEN
Cleft lip and palate are types of craniofacial birth defects that affect thousands of children worldwide each year. These conditions are sensitive topics of conversations, often affected by the stigma of physical birth deformities and cultural myths. This article reviews the pathophysiology of cleft lip and palate, and describes the traditional management of patients with oral-facial clefts, including the extensive supportive care and an interprofessional team or cleft team approach that extends beyond the surgical correction.
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Labio Leporino , Fisura del Paladar , Lactancia Materna , Labio Leporino/embriología , Labio Leporino/etiología , Labio Leporino/prevención & control , Labio Leporino/cirugía , Fisura del Paladar/embriología , Fisura del Paladar/etiología , Fisura del Paladar/prevención & control , Fisura del Paladar/cirugía , Ácido Fólico/administración & dosificación , Humanos , Lactante , Grupo de Atención al Paciente , Cuidados Preoperatorios , Factores de RiesgoRESUMEN
2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) effectively induces cleft palate at increased doses, but its mechanism of involvement is unclear, and arguments have examined palatal shelf contact and/or fusion failure. The role of different types of cells constituting palatal skulls remains elusive regarding TCDD dosage. No reports have simultaneously compared the biological behaviors of TCDD- induced mesenchymal and epithelial cells in vitro. This study employed primary epithelial and mesenchymal cells as models in vitro to explore proliferation, migration, apoptosis and epithelial-to-mesenchymal transition with two different doses of TCDD (10 nmol/L, 100 nmol/L), contrasted with a control group without TCDD. Interestingly, we found the EMT process of primary palatal epithelial cells occurred automatically in vitro without helping bilateral palatal contact. The results showed that, with the low dose of TCDD, transformation of epithelial cells to mesenchymal cells was inhibited, and mesenchymal cell proliferation and migration were promoted. At high doses, mesenchymal cells decreased, preventing palate development, uprising and contact, while the EMT of epithelial cells decreased. Regardless of dose of TCDD, no impact on migration and apoptosis of epithelial cells was noted, but there was increased apoptosis of mesenchymal cell in a dose-dependent manner.
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Desarrollo Embrionario/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Células Epiteliales/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Hueso Paladar/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Animales , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fisura del Paladar/inducido químicamente , Fisura del Paladar/embriología , Fisura del Paladar/patología , Relación Dosis-Respuesta a Droga , Células Epiteliales/patología , Femenino , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos C57BL , Hueso Paladar/embriología , Hueso Paladar/patología , Embarazo , Efectos Tardíos de la Exposición Prenatal/patologíaRESUMEN
Cleft lip is one of the most common human birth defects. However, there remain a limited number of mouse models of cleft lip that can be leveraged to characterize the genes and mechanisms that cause this disorder. Crosstalk between epithelial and mesenchymal cells underlies formation of the face and palate, but the basic molecular events mediating this crosstalk remain poorly understood. We previously demonstrated that mice lacking the epithelial-specific splicing factor Esrp1 have fully penetrant bilateral cleft lip and palate. In this study, we further investigated the mechanisms leading to cleft lip as well as cleft palate in both existing and new Esrp1 mutant mouse models. These studies included a detailed transcriptomic analysis of changes in ectoderm and mesenchyme in Esrp1-/- embryos during face formation. We identified altered expression of genes previously implicated in cleft lip and/or palate, including components of multiple signaling pathways. These findings provide the foundation for detailed investigations using Esrp1 mutant disease models to examine gene regulatory networks and pathways that are essential for normal face and palate development - the disruption of which leads to orofacial clefting in human patients.
Asunto(s)
Labio Leporino/patología , Fisura del Paladar/patología , Epitelio/patología , Mesodermo/patología , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Empalme Alternativo/genética , Animales , Proliferación Celular , Labio Leporino/embriología , Labio Leporino/genética , Fisura del Paladar/embriología , Fisura del Paladar/genética , Ectodermo/embriología , Ectodermo/metabolismo , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Epitelio/embriología , Cara , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Mesodermo/embriología , Ratones Noqueados , Organogénesis/genética , Hueso Paladar/embriología , Hueso Paladar/patologíaRESUMEN
In this study, we investigated the role of the transcription factor Six2 in palate development. Six2 was selected using the SysFACE tool to predict genes from the 2p21 locus, a region associated with clefting in humans by GWAS, that are likely to be involved in palatogenesis. We functionally validated the predicted role of Six2 in palatogenesis by showing that 22% of Six2 null embryos develop cleft palate. Six2 contributes to palatogenesis by promoting mesenchymal cell proliferation and regulating bone formation. The clefting phenotype in Six2-/- embryos is similar to Pax9 null embryos, so we examined the functional relationship of these two genes. Mechanistically, SIX2 binds to a PAX9 5' upstream regulatory element and activates PAX9 expression. In addition, we identified a human SIX2 coding variant (p.Gly264Glu) in a proband with cleft palate. We show this missense mutation affects the stability of the SIX2 protein and leads to decreased PAX9 expression. The low penetrance of clefting in the Six2 null mouse combined with the mutation in one patient with cleft palate underscores the potential combinatorial interactions of other genes in clefting. Our study demonstrates that Six2 interacts with the developmental gene regulatory network in the developing palate.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Factor de Transcripción PAX9/genética , Factores de Transcripción/metabolismo , Animales , Fisura del Paladar/embriología , Fisura del Paladar/genética , Anomalías Craneofaciales/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Genes Homeobox , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Proteínas del Tejido Nervioso/metabolismo , Osteogénesis , Factor de Transcripción PAX9/metabolismo , Factores de Transcripción Paired Box , Hueso Paladar/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
The mammalian secondary palate forms from two shelves of mesenchyme sheathed in a single-layered epithelium. These shelves meet during embryogenesis to form the midline epithelial seam (MES). Failure of MES degradation prevents mesenchymal confluence and results in a cleft palate. Previous studies indicated that MES cells undergo features of epithelial-to-mesenchymal transition (EMT) and may become migratory as part of the fusion mechanism. To detect MES cell movement over the course of fusion, we imaged the midline of fusing embryonic ephrin-B2/GFP mouse palates in real time using two-photon microscopy. These mice express an ephrin-B2-driven green fluorescent protein (GFP) that labels the palatal epithelium nuclei and persists in those cells through the time window necessary for fusion. We observed collective migration of MES cells toward the oral surface of the palatal shelf over 48 hr of imaging, and we confirmed histologically that the imaged palates had fused by the end of the imaged period. We previously reported that ephrin reverse signaling in the MES is required for palatal fusion. We therefore added recombinant EphA4/Fc protein to block this signaling in imaged palates. The blockage inhibited fusion, as expected, but did not change the observed migration of GFP-labeled cells. Thus, we uncoupled migration and fusion. Our data reveal that palatal MES cells undergo a collective, unidirectional movement during palatal fusion and that ephrin reverse signaling, though required for fusion, controls aspects of the fusion mechanism independent of migration.
Asunto(s)
Movimiento Celular/fisiología , Fisura del Paladar/embriología , Hueso Paladar/embriología , Animales , Células Epiteliales/fisiología , Transición Epitelial-Mesenquimal/fisiología , RatonesRESUMEN
OBJECTIVES: To describe a novel sign, the 'superimposed-line' sign, for early diagnosis of cleft of the fetal secondary palate on two-dimensional imaging of the vomeromaxillary junction in the midsagittal view. METHODS: This was a prospective evaluation of the superimposed-line sign using two-dimensional sonography (midsagittal view) in 9576 singleton fetuses referred for routine screening between 12 and 20 weeks of gestation. In this view, the vomer bone appears as a line superimposed on the distal two-thirds of the maxillary line, as the vomer fuses with the secondary palate in the midline. If there is a midline cleft of the secondary palate, the line formed by the palate is absent and hence only the vomer bone is visualized, creating a single line instead of the normal superimposed double line. Multiplanar three-dimensional (3D) views were assessed in cases in which the superimposed-line sign was absent. RESULTS: The superimposed line was absent in 17 fetuses with a cleft of the secondary palate that was confirmed by 3D evaluation. Of these, 13 had defects involving the premaxilla and four had an isolated cleft of the secondary palate. Postnatal confirmation was available in all cases. The sign was useful in ruling out cleft of the fetal secondary palate in 32 high-risk cases with a family history of cleft palate. The superimposed-line sign had a sensitivity of 89.5% in detecting cleft of the secondary palate. CONCLUSIONS: The superimposed-line sign is a new sonographic marker for evaluation of cleft of the fetal secondary palate; documentation of this sign proves the presence of both the palate and vomer in the midline. This marker can be demonstrated clearly in the late first trimester, allowing early diagnosis of secondary palatine cleft. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.
Asunto(s)
Fisura del Paladar/diagnóstico por imagen , Maxilar/embriología , Hueso Paladar/embriología , Ultrasonografía Prenatal/métodos , Vómer/embriología , Adulto , Biomarcadores/análisis , Fisura del Paladar/embriología , Diagnóstico Precoz , Femenino , Edad Gestacional , Humanos , Imagenología Tridimensional/métodos , Maxilar/diagnóstico por imagen , Hueso Paladar/diagnóstico por imagen , Embarazo , Estudios Prospectivos , Vómer/diagnóstico por imagen , Adulto JovenAsunto(s)
Aneuploidia , Trastornos de los Cromosomas/diagnóstico por imagen , Asesoramiento Genético/métodos , Atención Prenatal/métodos , Ultrasonografía Prenatal , Labio Leporino/diagnóstico por imagen , Labio Leporino/embriología , Fisura del Paladar/diagnóstico por imagen , Fisura del Paladar/embriología , Obstrucción Duodenal/diagnóstico por imagen , Obstrucción Duodenal/embriología , Femenino , Humanos , Atresia Intestinal/diagnóstico por imagen , Atresia Intestinal/embriología , Embarazo , Estudios RetrospectivosRESUMEN
OBJECTIVE: Cleft palate (CP) is a congenital birth defect caused by the failure of palatal fusion. Little is known about the potential role of DNA methylation in the pathogenesis of CP. This study aimed to explore the potential role of DNA methylation in the mechanism of CP. METHODOLOGY: We established an all-trans retinoic acid (ATRA)-induced CP model in C57BL/6J mice and used methylation-dependent restriction enzymes (MethylRAD, FspEI) combined with high-throughput sequencing (HiSeq X Ten) to compare genome-wide DNA methylation profiles of embryonic mouse palatal tissues, between embryos from ATRA-treated vs. untreated mice, at embryonic gestation day 14.5 (E14.5) (n=3 per group). To confirm differentially methylated levels of susceptible genes, real-time quantitative PCR (qPCR) was used to correlate expression of differentially methylated genes related to CP. RESULTS: We identified 196 differentially methylated genes, including 17,298 differentially methylated CCGG sites between ATRA-treated vs. untreated embryonic mouse palatal tissues (P<0.05, log2FC>1). The CP-related genes Fgf16 (P=0.008, log2FC=1.13) and Tbx22 (P=0.011, log2FC=1.64,) were hypermethylated. Analysis of Fgf16 and Tbx22, using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), identified 3 GO terms and 1 KEGG pathway functionally related to palatal fusion. The qPCR showed that changes in expression level negatively correlated with methylation levels. CONCLUSIONS: Taken together, these results suggest that hypermethylation of Fgf16 and Tbx22 is associated with decreased gene expression, which might be responsible for developmental failure of palatal fusion, eventually resulting in the formation of CP.
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Fisura del Paladar/genética , Metilación de ADN , Factores de Crecimiento de Fibroblastos/genética , Expresión Génica , Proteínas de Dominio T Box/genética , Animales , Fisura del Paladar/embriología , Fisura del Paladar/patología , Femenino , Factores de Crecimiento de Fibroblastos/análisis , Masculino , Ratones Endogámicos C57BL , Dominios y Motivos de Interacción de Proteínas , Reacción en Cadena en Tiempo Real de la Polimerasa , Valores de Referencia , Análisis de Secuencia de ADN , Proteínas de Dominio T Box/análisisRESUMEN
OBJECTIVE: To investigate the correlation between down-regulation of miR-381-3p and inhibition of osteogenic differentiation of mouse embryonic palatal mesenchymal (MEPM) cells in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cleft palate of fetal mice. METHODS: Thirty-two pregnant mice were randomly divided into TCDD group and control group, 16 in each group. On embryonic day 10.5 (E10.5), the pregnant mice in TCDD group were orally administrated with TCDD at dosage of 28 µg/kg, while the pregnant mice in control group received equivalent corn oil. The pregnant mice in each group were sacrificed on E13.5 and E14.5, fetal palates were collected for analysis. The expression of miR-381-3p was detected by real-time fluorescent quantitative PCR and the protein expressions of runt- related transcription factor 2 (RUNX2) and osteopontin (OPN) were detected by Western blot. MEPM cells were extracted from fetal palates on E14.5 in control group and passaged. The 3rd passage cells were cultured with TCDD at dosage of 10 nmol/L for 0, 0.5, 1, 2, and 3 days. The expression of miR-381-3p was detected after 0, 0.5, 1, 2, and 3 days and the protein expressions of RUNX2 and OPN were detected after 0, 1, 2, and 3 days. Then, the 3rd passage cells were divided into 4 groups. The MEPM cells were transfected with miR-381-3p inhibitor (inhibitor group), NC inhibitor (NC inhibitor group) and miR-381-3p mimics (mimics group), NC mimics (NC mimics group) for 48 hours, respectively. And the expressions of miR-381-3p and the protein expressions of RUNX2 and OPN were detected. RESULTS: On E13.5 and E14.5, 96 fetal mice in control group and 92 in TCDD group were obtained. The bilateral palates contacted in control group on E14.5, and a gap between the bilateral palates existed in TCDD group. On E13.5 and E14.5, the relative expressions of miR-381-3p and RUNX2 and OPN proteins were significant lower in TCDD group than in control group ( P<0.05). The relative expression of miR-381-3p at 0.5 and 1 day after TCDD treatment of MEPM cells were significantly lower than that at 0 day ( P<0.05); then, the relative expressions at 2 and 3 days significantly increased, showing no significant difference when compared with that at 0 day ( P>0.05). The relative expressions of RUNX2 and OPN proteins at 1, 2, and 3 days were significantly lower than that at 0 day ( P<0.05). The relative expressions of miR-381-3p and RUNX2 and OPN proteins significantly lower in inhibitor group than in NC inhibitor group ( P<0.05) and higher in mimics group than in NC mimics group ( P<0.05). CONCLUSION: Down-regulation of miR-381-3p expression may be associated with inhibition of osteogenic differentiation of MEPM cells in TCDD-induced cleft palate of fetal mice.
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Fisura del Paladar , Regulación hacia Abajo , MicroARNs , Dibenzodioxinas Policloradas , Animales , Fisura del Paladar/inducido químicamente , Fisura del Paladar/embriología , Fisura del Paladar/genética , Fisura del Paladar/fisiopatología , Femenino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Osteogénesis/genética , Hueso Paladar/fisiopatología , EmbarazoRESUMEN
The aim of the present study was to determine the association between maternal metabolism and development of the fetal palate, and to suggest a potential noninvasive prenatal diagnostic method for fetal cleft palate (CP). Dexamethasone (DXM) was used to create a CP mouse model. A 9.4Tesla (T) magnetic resonance spectroscopy (MRS) imager was used to measure an array of metabolites in the maternal serum, placental tissue, amniotic fluid and fetal palates. Multivariate statistical analysis was performed using SIMCAP 14.1 software. Following DXM treatment, variations were detected in multiple metabolites in the female mice and their fetuses based on 9.4T MRS. It was indicated that in the experimental group during CP formation, leucine, valine, creatine, acetate and citrate levels in the palatal tissue were lower, whereas lactate, alanine, proline/inositol and glutamatecontaining metabolite levels were higher, compared with the levels in the control group. In placental tissue and amniotic fluid, succinate and choline levels were lower in the experimental group. The relative concentrations of cholesterol and lipids in palatal tissues from mice treated with DXM were higher compared with the concentrations in tissues from mice in the control group, with the exception of (CH2)n lipids. In the placental tissue, the alteration in cholesterol level exhibited the opposite trend. Lipid levels for the different lipid forms varied and most of them were unsaturated lipids.