RESUMO
Clefting of the secondary palate is one of the most common congenital anomalies, and the multiple corrective surgeries that individuals with isolated cleft palate undergo are associated with major costs and morbidities. Secondary palate development is a complex, multistep process that includes the elevation of the palatal shelves from a vertical to horizontal position, a process that is not well understood. The Hippo signaling cascade is a mechanosensory pathway that regulates morphogenesis, homeostasis, and regeneration by controlling cell proliferation, apoptosis, and differentiation, primarily via negative regulation of the downstream effectors, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). We deleted Yap/Taz throughout the palatal shelf mesenchyme as well as specifically in the posterior palatal shelf mesenchyme, using the Osr2Cre and Col2Cre drivers, respectively, which resulted in palatal shelf elevation delay and clefting of the secondary palate. In addition, the deletion resulted in undersized bones of the secondary palate. We next determined downstream targets of YAP/TAZ in the posterior palatal shelves, which included Ibsp and Phex, genes involved in mineralization, and Loxl4, which encodes a lysyl oxidase that catalyzes collagen crosslinking. Ibsp, Phex, and Loxl4 were expressed at decreased levels in the ossification region in the posterior palatal shelf mesenchyme upon deletion of Yap/Taz. Furthermore, collagen levels were decreased specifically in the same region prior to elevation. Thus, our data suggest that YAP/TAZ may regulate collagen crosslinking in the palatal shelf mesenchyme, thus controlling palatal shelf elevation, as well as mineralization of the bones of the secondary palate.
Assuntos
Fissura Palatina , Palato , Animais , Fissura Palatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Morfogênese , OsteogêneseRESUMO
In humans, ankyloglossia and cleft palate are common congenital craniofacial anomalies, and these are regulated by a complex gene regulatory network. Understanding the genetic underpinnings of ankyloglossia and cleft palate will be an important step toward rational treatment of these complex anomalies. We inactivated the Sry (sex-determining region Y)-box 2 (Sox2) gene in the developing oral epithelium, including the periderm, a transient structure that prevents abnormal oral adhesions during development. This resulted in ankyloglossia and cleft palate with 100% penetrance in embryos examined after embryonic day 14.5. In Sox2 conditional knockout embryos, the oral epithelium failed to differentiate, as demonstrated by the lack of keratin 6, a marker of the periderm. Further examination revealed that the adhesion of the tongue and mandible expressed the epithelial markers E-Cad and P63. The expanded epithelia are Sox9-, Pitx2-, and Tbx1-positive cells, which are markers of the dental epithelium; thus, the dental epithelium contributes to the development of oral adhesions. Furthermore, we found that Sox2 is required for palatal shelf extension, as well as for the formation of palatal rugae, which are signaling centers that regulate palatogenesis. In conclusion, the deletion of Sox2 in oral epithelium disrupts palatal shelf extension, palatal rugae formation, tooth development, and periderm formation. The periderm is required to inhibit oral adhesions and ankyloglossia, which is regulated by Sox2. In addition, oral adhesions occur through an expanded dental epithelial layer that inhibits epithelial invagination and incisor development. This process may contribute to dental anomalies due to ankyloglossia.
Assuntos
Fissura Palatina , Fissura Palatina/genética , Epitélio , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mucosa Bucal , Palato , Fatores de Transcrição SOXB1/genética , Transdução de SinaisRESUMO
Rare mutations in IRF6 and GRHL3 cause Van der Woude syndrome, an autosomal dominant orofacial clefting disorder. Common variants in IRF6 and GRHL3 also contribute risk for isolated orofacial clefting. Similarly, variants within genes that encode receptor tyrosine kinase (RTK) signaling components, including members of the FGF pathway, EPHA3 and SPRY2, also contribute risk for isolated orofacial clefting. In the mouse, loss of Irf6 or perturbation of Fgf signaling leads to abnormal oral epithelial adhesions and cleft palate. Oral adhesions can result from a disruption of periderm formation. Here, we find that IRF6 and SPRY4 signaling interact in periderm function. We crossed Irf6 heterozygous ( Irf6+/-) mice with transgenic mice that express Spry4 in the basal epithelial layer ( TgKRT14::Spry4). While embryos with either of these mutations can have abnormal oral adhesions, using a new quantitative assay, we observed a nonadditive effect of abnormal oral epithelial adhesions in the most severely affected double mutant embryos ( Irf6+/-;TgKRT14::Spry4). At the molecular level, the sites of abnormal oral adhesions maintained periderm-like cells that express keratin 6, but we observed abnormal expression of GRHL3. Together, these data suggest that Irf6 and RTK signaling interact in regulating periderm differentiation and function, as well as provide a rationale to screen for epistatic interactions between variants in IRF6 and RTK signaling pathway genes in human orofacial clefting populations.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Fatores Reguladores de Interferon/genética , Proteínas do Tecido Nervoso/genética , Aderências Teciduais/genética , Anormalidades Múltiplas/embriologia , Anormalidades Múltiplas/genética , Animais , Fenda Labial/embriologia , Fissura Palatina/embriologia , Cistos/embriologia , Cistos/genética , Modelos Animais de Doenças , Anormalidades Maxilomandibulares/embriologia , Anormalidades Maxilomandibulares/genética , Lábio/anormalidades , Lábio/embriologia , Camundongos , Camundongos Transgênicos , Anormalidades da Boca/embriologia , Anormalidades da Boca/genética , Mutação , Fenótipo , Transdução de Sinais , Aderências Teciduais/embriologiaRESUMO
OBJECTIVES: The RASopathies are a group of syndromes that have in common germline mutations in genes that encode components of the Ras/mitogen-activated protein kinase (MAPK) pathway and have been a focus of study to understand the role of this pathway in development and disease. These syndromes include Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML or LEOPARD syndrome), neurofibromatosis type 1 (NF1), Costello syndrome (CS), cardio-facio-cutaneous (CFC) syndrome, neurofibromatosis type 1-like syndrome (NFLS or Legius syndrome) and capillary malformation-arteriovenous malformation syndrome (CM-AVM). These disorders affect multiple systems, including the craniofacial complex. Although the craniofacial features have been well described and can aid in clinical diagnosis, the dental phenotypes have not been analysed in detail for each of the RASopathies. In this review, we summarize the clinical features of the RASopathies, highlighting the reported craniofacial and dental findings. METHODS: Review of the literature. RESULTS: Each of the RASopathies reviewed, caused by mutations in genes that encode different proteins in the Ras pathway, have unique and overlapping craniofacial and dental characteristics. CONCLUSIONS: Careful description of craniofacial and dental features of the RASopathies can provide information for dental clinicians treating these individuals and can also give insight into the role of Ras signalling in craniofacial development.
Assuntos
Anormalidades Craniofaciais/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas ras/genética , Malformações Arteriovenosas/genética , Manchas Café com Leite/genética , Capilares/anormalidades , Síndrome de Costello , Displasia Ectodérmica/genética , Fácies , Insuficiência de Crescimento/genética , Mutação em Linhagem Germinativa , Cardiopatias Congênitas/genética , Humanos , Síndrome LEOPARD , Neurofibromatose 1/genética , Síndrome de Noonan/genética , Mancha Vinho do Porto/genéticaRESUMO
The role of Ras signaling during tooth development is poorly understood. Ras proteins-which are activated by many upstream pathways, including receptor tyrosine kinase cascades-signal through multiple effectors, such as the mitogen-activated protein kinase (MAPK) and PI3K pathways. Here, we utilized the mouse incisor as a model to study how the MAPK and PI3K pathways regulate dental epithelial stem cells and amelogenesis. The rodent incisor-which grows continuously throughout the life of the animal due to the presence of epithelial and mesenchymal stem cells-provides a model for the study of ectodermal organ renewal and regeneration. Utilizing models of Ras dysregulation as well as inhibitors of the MAPK and PI3K pathways, we found that MAPK and PI3K regulate dental epithelial stem cell activity, transit-amplifying cell proliferation, and enamel formation in the mouse incisor.
Assuntos
Amelogênese/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Proteínas ras/metabolismo , Animais , Benzamidas/farmacologia , Proliferação de Células , Classe I de Fosfatidilinositol 3-Quinases , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Imunofluorescência , Incisivo , Indazóis/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Animais , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Sulfonamidas/farmacologiaRESUMO
Social interaction is a fundamental behavior in all animal species, but the developmental timing of the social neural circuit formation and the cellular and molecular mechanisms governing its formation are poorly understood. We generated a mouse model with mutations in two Disheveled genes, Dvl1 and Dvl3, that displays adult social and repetitive behavioral abnormalities associated with transient embryonic brain enlargement during deep layer cortical neuron formation. These phenotypes were mediated by the embryonic expansion of basal neural progenitor cells (NPCs) via deregulation of a ß-catenin/Brn2/Tbr2 transcriptional cascade. Transient pharmacological activation of the canonical Wnt pathway during this period of early corticogenesis rescued the ß-catenin/Brn2/Tbr2 transcriptional cascade and the embryonic brain phenotypes. Remarkably, this embryonic treatment prevented adult behavioral deficits and partially rescued abnormal brain structure in Dvl mutant mice. Our findings define a mechanism that links fetal brain development and adult behavior, demonstrating a fetal origin for social and repetitive behavior deficits seen in disorders such as autism.
Assuntos
Transtorno de Movimento Estereotipado/genética , Transtorno de Movimento Estereotipado/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Comportamento Animal , Encéfalo/embriologia , Encéfalo/metabolismo , Encéfalo/fisiologia , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fatores do Domínio POU/metabolismo , Fatores do Domínio POU/fisiologia , Fosfoproteínas/genética , Transdução de Sinais/fisiologia , Comportamento Estereotipado/fisiologia , Proteínas com Domínio T/metabolismo , Proteínas com Domínio T/fisiologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , beta Catenina/fisiologiaRESUMO
Stem cells from the apical papilla (SCAPs) are important for the formation and regeneration of root dentin. Here, we examined the expression of Notch signaling components in SCAPs and investigated crosstalk between microRNA miR-34aand Notch signaling during cell differentiation. We found that human SCAPs express NOTCH2, NOTCH3, JAG2, DLL3, and HES1, and we tested the relationship between Notch signaling and both cell differentiation and miR-34a expression. NOTCH activation in SCAPs inhibited cell differentiation and up-regulated the expression of miR-34a, whereas miR-34a inhibited Notch signaling in SCAPs by directly targeting the 3'UTR of NOTCH2 and HES1 mRNA and suppressing the expression of NOTCH2, N2ICD, and HES1. DSPP, RUNX2, OSX, and OCN expression was consequently up-regulated. Thus, Notch signaling in human SCAPs plays a vital role in maintenance of these cells. miR-34a interacts with Notch signaling and promotes both odontogenic and osteogenic differentiation of SCAPs.
Assuntos
MicroRNAs/fisiologia , Receptor Cross-Talk/fisiologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Ápice Dentário/citologia , Regiões 3' não Traduzidas/genética , Adolescente , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Criança , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Jagged-2 , Proteínas de Membrana/genética , Odontogênese/genética , Osteocalcina/genética , Osteogênese/genética , Fosfoproteínas/genética , Receptor Notch2/genética , Receptor Notch3 , Receptores Notch/genética , Proteínas Repressoras/genética , Sialoglicoproteínas/genética , Fator de Transcrição Sp7 , Fatores de Transcrição HES-1 , Fatores de Transcrição/genética , Adulto JovemRESUMO
Cardio-facio-cutaneous syndrome (CFC) is a RASopathy that is characterized by craniofacial, dermatologic, gastrointestinal, ocular, cardiac, and neurologic anomalies. CFC is caused by activating mutations in the Ras/mitogen-activated protein kinase (MAPK) signaling pathway that is downstream of receptor tyrosine kinase (RTK) signaling. RTK signaling is known to play a central role in craniofacial and dental development, but to date, no studies have systematically examined individuals with CFC to define key craniofacial and dental features. To fill this critical gap in our knowledge, we evaluated the craniofacial and dental phenotype of a large cohort (n = 32) of CFC individuals who attended the 2009 and 2011 CFC International Family Conferences. We quantified common craniofacial features in CFC which include macrocephaly, bitemporal narrowing, convex facial profile, and hypoplastic supraorbital ridges. In addition, there is a characteristic dental phenotype in CFC syndrome that includes malocclusion with open bite, posterior crossbite, and a high-arched palate. This thorough evaluation of the craniofacial and dental phenotype in CFC individuals provides a step forward in our understanding of the role of RTK/MAPK signaling in human craniofacial development and will aid clinicians who treat patients with CFC.
Assuntos
Anormalidades Craniofaciais/patologia , Displasia Ectodérmica/patologia , Insuficiência de Crescimento/patologia , Cardiopatias Congênitas/patologia , Anormalidades Dentárias/patologia , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Displasia Ectodérmica/genética , Fácies , Insuficiência de Crescimento/genética , Feminino , Genótipo , Cardiopatias Congênitas/genética , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Masculino , Fenótipo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais/genética , Síndrome , Adulto Jovem , Proteínas ras/genéticaRESUMO
The temporomandibular joint (TMJ) is a specialized synovial joint essential for the function of the mammalian jaw. The main components of the TMJ are the mandibular condyle, the glenoid fossa of the temporal bone, and a fibrocartilagenous disc interposed between them. The genetic program for the development of the TMJ remains poorly understood. Here we show the crucial role of sprouty (Spry) genes in TMJ development. Sprouty genes encode intracellular inhibitors of receptor tyrosine kinase (RTK) signaling pathways, including those triggered by fibroblast growth factors (Fgfs). Using in situ hybridization, we show that Spry1 and Spry2 are highly expressed in muscles attached to the TMJ, including the lateral pterygoid and temporalis muscles. The combined inactivation of Spry1 and Spry2 results in overgrowth of these muscles, which disrupts normal development of the glenoid fossa. Remarkably, condyle and disc formation are not affected in these mutants, demonstrating that the glenoid fossa is not required for development of these structures. Our findings demonstrate the importance of regulated RTK signaling during TMJ development and suggest multiple skeletal origins for the fossa. Notably, our work provides the evidence that the TMJ condyle and disc develop independently of the mandibular fossa.
Assuntos
Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Proteínas de Membrana/genética , Fosfoproteínas/genética , Articulação Temporomandibular/embriologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antimetabólitos , Apoptose/genética , Bromodesoxiuridina , Caspase 3/análise , Proliferação de Células , Fatores de Crescimento de Fibroblastos/genética , Inativação Gênica , Idade Gestacional , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Côndilo Mandibular/embriologia , Camundongos , Camundongos Knockout , Mutação/genética , Proteínas Serina-Treonina Quinases , Músculos Pterigoides/embriologia , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/genética , Osso Temporal/embriologia , Músculo Temporal/embriologia , Disco da Articulação Temporomandibular/embriologia , Microtomografia por Raio-XRESUMO
Interstitial deletions of the long arm of chromosome 6 are relatively rare, with fewer than 100 cases reported. Phenotypic variation is in large part due to differences in size and location of the segmental aneuploidy. We report three new patients with interstitial deletions of chromosome 6q defined at the molecular level by array comparative genomic hybridization (array CGH). In two of three cases, the molecular breakpoints differed from those indicated by conventional karyotyping, demonstrating the enhanced resolution of array CGH. Two patients had minimal deletions of 6 and 8.8 Mb involving 6q16.2-->q21, and the third patient had a deletion of 11.3 Mb spanning 6q15-->q21. All three had developmental delay, craniofacial dysmorphology, and functional eye disorders, suggesting that genes affecting brain and craniofacial development are located in 6q16.2-->q21, the deleted region common to all three patients. Furthermore, gene(s) for discordant phenotypic features, such as central diabetes insipidus, may reside at 6q15, the monosomic region unique to patient 3. All three cases described here showed loss of paternal alleles within the deleted segment, providing further evidence of the predominantly paternal origin for 6q deletions and rearrangements.
Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Deleção Cromossômica , Cromossomos Humanos Par 6 , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Adolescente , Aneuploidia , Criança , Análise Citogenética , Feminino , Genótipo , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , FenótipoRESUMO
Prader-Willi syndrome (PWS) is caused by lack of expression of paternally inherited genes on chromosome 15q11-->15q13. Most cases result from microdeletions in proximal chromosome 15q. The remainder results from maternal uniparental disomy of chromosome 15, imprinting center defects, and rarely from balanced or unbalanced chromosome rearrangements involving chromosome 15. We report a patient with multiple congenital anomalies, including craniofacial dysmorphology, microcephaly, bilateral cryptorchidism, and developmental delay. Cytogenetic analysis showed a de novo 45,XY,der(5)t(5;15)(p15.2;q13), -15 karyotype. In effect, the proband had monosomies of 5p15.2-->pter and 15pter-->15q13. Methylation polymerase chain reaction analysis of the promoter region of the SNRPN gene showed only the maternal allele, consistent with the PWS phenotype. The proband's expanded phenotype was similar to other patients who have PWS as a result of unbalanced translocations and likely reflects the contribution of the associated monosomy. Array comparative genomic hybridization (array CGH) confirmed deletions of both distal 5p and proximal 15q and provided more accurate information as to the size of the deletions and the molecular breakpoints. This case illustrates the utility of array CGH in characterizing complex constitutional structural chromosome abnormalities at the molecular level.
Assuntos
Síndrome de Prader-Willi/genética , Translocação Genética/genética , Deleção Cromossômica , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 5/genética , Cromossomos Humanos Par 5/ultraestrutura , Ilhas de CpG/genética , Metilação de DNA , Humanos , Hibridização in Situ Fluorescente , Recém-Nascido , Cariotipagem , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Síndrome de Prader-Willi/diagnósticoRESUMO
Two site-directed mutants of isocitrate dehydrogenase (IDH) of Escherichia coli have been studied by site-directed mutagenesis kinetic and structural studies. Substitution of phenylalanine for tyrosine at position 160 (Y160F) showed 0.4% of the kcat of wild-type with isocitrate as substrate, while the Km for isocitrate remained unchanged. When the postulated intermediate, oxalosuccinate, was enzymatically decarboxylated, Y160F showed a higher kcat and a similar Km to the wild type values. The rate of reduction of oxalosuccinate to isocitrate by the Y160F mutant was greatly decreased relative to the wild-type. Substitution of methionine for lysine at position 230 decreased kcat to 1.1% of that of the wild-type and Km increased by a factor of 500-600. The decarboxylation of oxalosuccinate was undetectable for the K230M mutant. The structure of the site-directed mutants of IDH with and without a bound complex of isocitrate and Mg2+ was solved at 2.5 A resolution and compared by difference mapping against previously determined enzyme structures. The structural studies show that (i) the overall protein-folding side chain conformations and active sites of both mutants are isomorphous with wild-type enzyme, (ii) isocitrate and magnesium bind to both enzyme mutants with the same relative conformation and binding interactions as wild-type enzyme, and (iii) the mutated side chains (Phe 160 and Met 230) are positioned for catalysis in a similar conformation as that observed for the wild-type enzyme. Hence, the alteration of the side chain functional groups is directly related to the loss of enzyme activity. Possible roles of the active site tyrosine and lysine are discussed.