RESUMEN
BACKGROUND: Fibroblast growth factors (Fgfs) are required for survival and organ formation during embryogenesis. Fgfs often execute their functions redundantly. Previous analysis of Fgf3 mutants revealed effects on inner ear formation and embryonic survival with incomplete penetrance. RESULTS: Here, we show that presence of a neomycin resistance gene (neo) replacing the Fgf3 coding region leads to reduced survival during embryogenesis and an increased penetrance of inner ear defects. Fgf3neo/neo mutants showed reduced expression of Fgf4, which is positioned in close proximity to the Fgf3 locus in the mouse genome. Conditional inactivation of Fgf4 during inner ear development on a Fgf3 null background using Fgf3/4 cis mice revealed a redundant requirement between these Fgfs during otic placode induction. In contrast, inactivation of Fgf3 and Fgf4 in the pharyngeal region where both Fgfs are also co-expressed using a Foxg1-Cre driver did not affect development of the pharyngeal arches. However, these mutants showed reduced perinatal survival. CONCLUSIONS: These results highlight the importance of Fgf signaling during development. In particular, different members of the Fgf family act redundantly to guarantee inner ear formation and embryonic survival.
Asunto(s)
Oído Interno , Factores de Crecimiento de Fibroblastos , Animales , Ectodermo/metabolismo , Femenino , Factor 3 de Crecimiento de Fibroblastos/genética , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Factor 4 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción Forkhead/genética , Ratones , Familia de Multigenes , Proteínas del Tejido Nervioso/genética , EmbarazoRESUMEN
Pan-otic CRE drivers enable gene regulation throughout the otic placode lineage, comprising the inner ear epithelium and neurons. However, intersection of extra-otic gene-of-interest expression with the CRE lineage can compromise viability and impede auditory analyses. Furthermore, extant pan-otic CREs recombine in auditory and vestibular brain nuclei, making it difficult to ascribe resulting phenotypes solely to the inner ear. We have previously identified Slc26a9 as an otic placode-specific target of the FGFR2b ligands FGF3 and FGF10. We show here that Slc26a9 is otic specific through E10.5, but is not required for hearing. We targeted P2ACre to the Slc26a9 stop codon, generating Slc26a9P2ACre mice, and observed CRE activity throughout the otic epithelium and neurons, with little activity evident in the brain. Notably, recombination was detected in many FGFR2b ligand-dependent epithelia. We generated Fgf10 and Fgf8 conditional mutants, and activated an FGFR2b ligand trap from E17.5 to P3. In contrast to analogous mice generated with other pan-otic CREs, these were viable. Auditory thresholds were elevated in mutants, and correlated with cochlear epithelial cell losses. Thus, Slc26a9P2ACre provides a useful complement to existing pan-otic CRE drivers, particularly for postnatal analyses.
Asunto(s)
Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Antiportadores/genética , Antiportadores/metabolismo , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factor 3 de Crecimiento de Fibroblastos/genética , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Factor 8 de Crecimiento de Fibroblastos/genética , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Ratones , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismoRESUMEN
Thalamus is an important sensory relay station: afferent sensory information, except olfactory signals, is transmitted by thalamocortical axons (TCAs) to the cerebral cortex. The pathway choice of TCAs depends on diverse diffusible or substrate-bound guidance cues in the environment. Not only classical guidance cues (ephrins, slits, semaphorins, and netrins), morphogens, which exerts patterning effects during early embryonic development, can also help axons navigate to their targets at later development stages. Here, expression analyses reveal that morphogen Fibroblast growth factor (FGF)-3 is expressed in the chick ventral diencephalon, hypothalamus, during the pathfinding of TCAs. Then, using in vitro analyses in chick explants, we identify a concentration-dependent effect of FGF3 on thalamic axons: attractant 100 ng/mL FGF3 transforms to a repellent at high concentration 500 ng/mL. Moreover, inhibition of FGF3 guidance functions indicates that FGF3 signaling is necessary for the correct navigation of thalamic axons. Together, these studies demonstrate a direct effect for the member of FGF7 subfamily, FGF3, in the axonal pathfinding of TCAs.
Asunto(s)
Orientación del Axón/fisiología , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Hipotálamo/metabolismo , Vías Nerviosas/embriología , Animales , Corteza Cerebral/embriología , Embrión de Pollo , Tálamo/embriologíaRESUMEN
Previous studies indicated that the elevated mesenchymal Wnt/ß-catenin signaling deprived dental mesenchyme of odontogenic fate. By utilizing ex vivo or pharmacological approaches, Wnt/ß-catenin signaling in the developing dental mesenchyme was suggested to suppress the odontogenic fate by disrupting the balance between Axin2 and Runx2. In our study, the Osr2-creKI; Ctnnb1ex3f mouse was used to explore how mesenchymal Wnt/ß-catenin signaling suppressed the odontogenic fate in vivo. We found that all of the incisor and half of the molar germs of Osr2-creKI; Ctnnb1ex3fmice started to regress at E14.5 and almost disappeared at birth. The expression of Fgf3 and Msx1 was dramatically down-regulated in the E14.5 Osr2-creKI; Ctnnb1ex3f incisor and molar mesenchyme, while Runx2transcription was only diminished in incisor mesenchyme. Intriguingly, in the E14.5 Osr2-creKI; Ctnnb1ex3f incisor epithelium, the expression of Noggin was activated, while Shh was abrogated. Similarly, the Wnt and BMP antagonists, Ectodin and Noggin were also ectopically activated in the E14.5 Osr2-creKI; Ctnnb1ex3f molar epithelium. Recombination of E13.5 Osr2-creKI; Ctnnb1ex3f molar mesenchyme with E10.5 and E13.5 WT dental epithelia failed to develop tooth. Taken together, the mesenchymal Wnt/ß-catenin signaling resulted in the loss of odontogenic fate in vivo not only by directly suppressing odontogenic genes expression but also by inducing Wnt and BMP antagonists in dental epithelium.
Asunto(s)
Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Epitelio/metabolismo , Mesodermo/metabolismo , Boca/metabolismo , Diente/embriología , Vía de Señalización Wnt , Animales , Proliferación Celular , Supervivencia Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genotipo , Factor de Transcripción MSX1/metabolismo , Masculino , Ratones , Diente Molar/metabolismo , Odontogénesis , Organogénesis , Transducción de Señal , Proteína Wnt1/antagonistas & inhibidores , Proteína Wnt1/metabolismo , beta Catenina/metabolismoRESUMEN
Gastrointestinal stromal tumors (GIST) lacking mutations in KIT/PDGFRA or RAS pathways and retaining an intact SDH complex are usually referred to as KIT/PDGFRA/SDH/RAS-P WT GIST or more simply quadruple WT GIST (~5% of all GIST). Despite efforts made, no recurrent genetic event in quadruple WT GIST has been identified so far. To further investigate this disease, we performed high throughput copy number analysis on quadruple WT GIST specimens identifying a recurrent focal gain in band 11q13.3 (involving FGF3/FGF4) in 6/8 cases. This event was not found in the other molecular GIST subgroups. FGF3/FGF4 duplication was associated with high expression of FGF4, both at mRNA and protein level, a growth factor normally not expressed in adult tissues or in KIT/PDGFRA-mutated GIST. FGFR1 was found to be the predominant FGF receptor expressed and phosphorylation of AKT was detected, suggesting that a FGF4-FGFR1 autocrine loop could stimulate downstream signaling in quadruple WT GIST. Together with the recent reports of quadruple WT cases carrying FGFR1 activating alterations, these findings strengthen the hypothesis of a potential involvement of FGFR pathway deregulation in quadruple WT GIST, which may represent a rationale for novel therapeutic approaches.
Asunto(s)
Factor 4 de Crecimiento de Fibroblastos/genética , Neoplasias Gastrointestinales/genética , Tumores del Estroma Gastrointestinal/genética , Duplicación de Gen , Adulto , Anciano , Cromosomas Humanos Par 11/genética , Variaciones en el Número de Copia de ADN , Femenino , Factor 3 de Crecimiento de Fibroblastos/genética , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Factor 4 de Crecimiento de Fibroblastos/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-kit/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Succinato Deshidrogenasa/genética , Proteínas ras/genéticaRESUMEN
Morphogenesis of the inner ear epithelium requires coordinated deployment of several signaling pathways, and disruptions cause abnormalities of hearing and/or balance. The FGFR2b ligands FGF3 and FGF10 are expressed throughout otic development and are required individually for normal morphogenesis, but their prior and redundant roles in otic placode induction complicates investigation of subsequent combinatorial functions in morphogenesis. To interrogate these roles and identify new effectors of FGF3 and FGF10 signaling at the earliest stages of otic morphogenesis, we used conditional gene ablation after otic placode induction, and temporal inhibition of signaling with a secreted, dominant-negative FGFR2b ectodomain. We show that both ligands are required continuously after otocyst formation for maintenance of otic neuroblasts and for patterning and proliferation of the epithelium, leading to normal morphogenesis of both the cochlear and vestibular domains. Furthermore, the first genome-wide identification of proximal targets of FGFR2b signaling in the early otocyst reveals novel candidate genes for inner ear development and function.
Asunto(s)
Oído Interno/crecimiento & desarrollo , Oído Interno/metabolismo , Morfogénesis , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Linaje de la Célula , Proliferación Celular , Cóclea/crecimiento & desarrollo , Cóclea/metabolismo , Doxiciclina/farmacología , Femenino , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Ganglión/metabolismo , Regulación del Desarrollo de la Expresión Génica , Integrasas/metabolismo , Ligandos , Masculino , Ratones , Mutación/genética , Neuronas/citología , Neuronas/metabolismo , Factor de Transcripción PAX2/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Vestíbulo del Laberinto/crecimiento & desarrollo , Vestíbulo del Laberinto/metabolismoRESUMEN
BACKGROUND: Apert syndrome is characterized by craniosynostosis and bony syndactyly of the hands and feet. The cause of Apert syndrome is a single nucleotide substitution mutation (S252W or P253R) in fibroblast growth factor receptor 2 (FGFR2). Clinical experience suggests increased production of saliva by Apert syndrome patients, but this has not been formally investigated. FGFR2 signaling is known to regulate branching morphogenesis of the submandibular glands (SMGs). With the Apert syndrome mouse model (Ap mouse), we investigated the role of FGFR2 in SMGs and analyzed the SMG pathology of Apert syndrome. RESULTS: Ap mice demonstrated significantly greater SMG and sublingual gland (SMG/SLG complex) mass/body weight and percentage of parenchyma per unit area of the SMG compared with control mice. Furthermore, gene expression of Fgf1, Fgf2, Fgf3, Pdgfra, Pdgfrb, Mmp2, Bmp4, Lama5, Etv5, and Dusp6 was significantly higher in the SMG/SLG complex of Ap mice. FGF3 and BMP4 exhibited altered detection patterns. The numbers of macrophages were significantly greater in SMGs of Ap mice than in controls. Regarding functional evaluations of the salivary glands, no significant differences were observed. CONCLUSIONS: These results suggest that the gain-of-function mutation in FGFR2 in the SMGs of Ap mice enhances branching morphogenesis. Developmental Dynamics 247:1175-1185, 2018. © 2018 Wiley Periodicals, Inc.
Asunto(s)
Acrocefalosindactilia/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Glándula Submandibular/anomalías , Acrocefalosindactilia/patología , Animales , Proteína Morfogenética Ósea 4/metabolismo , Recuento de Células , Modelos Animales de Enfermedad , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Mutación con Ganancia de Función , Macrófagos/patología , Ratones , Morfogénesis , Glándula Submandibular/crecimiento & desarrolloRESUMEN
Genetic and epigenetic alterations observed at end stage OSCC formation could be considered as a consequence of cancer development and thus changes in normal or premalignant tissues which had been exposed to oral carcinogens such as Dibenzo[def,p]chrysene (DBP) may better serve as predictive biomarkers of disease development. Many types of DNA damage can induce epigenetic changes which can occur early and in the absence of evident morphological abnormalities. Therefore we used ERRBS to generate genome-scale, single-base resolution DNA methylomes from histologically normal oral tissues of mice treated with DBP under experimental conditions known to induce maximum DNA damage which is essential for the development of OSCC induced by DBP in mice. After genome-wide correction, 30 and 48 differentially methylated sites (DMS) were identified between vehicle control and DBP treated mice using 25% and 10% differences in methylation, respectively. RT-PCR was further performed to examine the expressions of nine selected genes. Among them, Fgf3, a gene frequently amplified in head and neck cancer, showed most prominent and significant gene expression change (2.4× increases), despite the hypomethylation of Fgf3 was identified at >10kb upstream of transcription start site. No difference was observed in protein expression between normal oral tissues treated with DBP or vehicle as examined by immunohistochemistry. Collectively, our results indicate that Fgf3 hypomethylation and gene overexpression, but not protein expression, occurred in the early stage of oral carcinogenesis induced by DBP. Thus, Fgf3 hypomethylation may serve as a potential biomarker for early detection of OSCC.
Asunto(s)
Benzopirenos/toxicidad , Biomarcadores de Tumor/metabolismo , Carcinógenos/toxicidad , Carcinoma de Células Escamosas/diagnóstico , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Neoplasias de la Boca/diagnóstico , Nicotiana/química , Animales , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Metilación de ADN , Detección Precoz del Cáncer , Femenino , Ratones , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The primary cilium is an outward projecting antenna-like organelle with an important role in bone mechanotransduction. The capacity to sense mechanical stimuli can affect important cellular and molecular aspects of bone tissue. Idiopathic scoliosis (IS) is a complex pediatric disease of unknown cause, defined by abnormal spinal curvatures. We demonstrate significant elongation of primary cilia in IS patient bone cells. In response to mechanical stimulation, these IS cells differentially express osteogenic factors, mechanosensitive genes, and signaling genes. Considering that numerous ciliary genes are associated with a scoliosis phenotype, among ciliopathies and knockout animal models, we expected IS patients to have an accumulation of rare variants in ciliary genes. Instead, our SKAT-O analysis of whole exomes showed an enrichment among IS patients for rare variants in genes with a role in cellular mechanotransduction. Our data indicates defective cilia in IS bone cells, which may be linked to heterogeneous gene variants pertaining to cellular mechanotransduction.
Asunto(s)
Cilios/genética , Regulación de la Expresión Génica , Mecanotransducción Celular/genética , Osteoblastos/metabolismo , Osteogénesis/genética , Escoliosis/genética , Adolescente , Huesos/metabolismo , Huesos/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Cilios/metabolismo , Cilios/patología , Exoma , Femenino , Factor 3 de Crecimiento de Fibroblastos/genética , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Osteoblastos/patología , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Cultivo Primario de Células , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Escoliosis/metabolismo , Escoliosis/patología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The inner ear is a morphologically complex sensory structure with auditory and vestibular functions. The developing otic epithelium gives rise to neurosensory and non-sensory elements of the adult membranous labyrinth. Extrinsic and intrinsic signals manage the patterning and cell specification of the developing otic epithelium by establishing lineage-restricted compartments defined in turn by differential expression of regulatory genes. FGF3 and FGF16 are excellent candidates to govern these developmental events. Using the chick inner ear, we show that Fgf3 expression is present in the borders of all developing cristae. Strong Fgf16 expression was detected in a portion of the developing vertical and horizontal pouches, whereas the cristae show weaker or undetected Fgf16 expression at different developmental stages. Concerning the rest of the vestibular sensory elements, both the utricular and saccular maculae were Fgf3 positive. Interestingly, strong Fgf16 expression delimited these Fgf16-negative sensory patches. The Fgf3-negative macula neglecta and the Fgf3-positive macula lagena were included within weakly Fgf16-expressing areas. Therefore, different FGF-mediated mechanisms might regulate the specification of the anterior (utricular and saccular) and posterior (neglecta and lagena) maculae. In the developing cochlear duct, dynamic Fgf3 and Fgf16 expression suggests their cooperation in the early specification and later cell differentiation in the hearing system. The requirement of Fgf3 and Fgf16 genes in endolymphatic apparatus development and neurogenesis are discussed. Based on these observations, FGF3 and FGF16 seem to be key signaling pathways that control the inner ear plan by defining epithelial identities within the developing otic epithelium.
Asunto(s)
Proteínas Aviares/metabolismo , Oído Interno/embriología , Oído Interno/metabolismo , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Animales , Pollos , Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
Odontogenesis is guided by a complex signaling cascade in which several molecules, including FGF2-4, ensure all dental groups development and specificity. Most of the data on odontogenesis derives from rodents, which does not have all dental groups. Didelphis albiventris is an opossum with the closest dentition to humans, and the main odontogenesis stages occur when the newborns are in the pouch. In this study, D. albiventris postnatals were used to characterize the main stages of their molars development; and also to establish FGF2, FGF3 and FGF4 expression pattern. D. albiventris postnatals were processed for histological and indirect immunoperoxidase analysis of the tooth germs. Our results revealed similar dental structures between D. albiventris and mice. However, FGF2, FGF3 and FGF4 expression patterns were observed in a larger number of dental structures, suggesting broader functions for these molecules in this opossum species. The knowledge of the signaling that determinates odontogenesis in an animal model with complete dentition may contribute to the development of therapies for the replacement of lost teeth in humans. This study may also contribute to the implementation of D. albiventris as model for Developmental Biology studies.
Asunto(s)
Didelphis/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Factor 4 de Crecimiento de Fibroblastos/metabolismo , Diente Molar/crecimiento & desarrollo , Odontogénesis , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Didelphis/crecimiento & desarrollo , Femenino , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 3 de Crecimiento de Fibroblastos/genética , Factor 4 de Crecimiento de Fibroblastos/genética , Ratones , Diente Molar/citología , Diente Molar/metabolismoRESUMEN
Studies have shown that fibroblast growth factor (Fgf) signalling is necessary for appendage regeneration, but its exact function and the ligands involved during regeneration have not yet been elucidated. Here, we performed comprehensive expression analyses and identified fgf20a and fgf3/10a as major Fgf ligands in the wound epidermis and blastema, respectively. To reveal the target cells and processes of Fgf signalling, we performed a transplantation experiment of mesenchymal cells that express the dominant-negative Fgf receptor 1 (dnfgfr1) under control of the heat-shock promoter. This mosaic knockdown analysis suggested that Fgf signalling is directly required for fin ray mesenchyme to form the blastema at the early pre-blastema stage and to activate the regenerative cell proliferation at a later post-blastema stage. These results raised the possibility that the early epidermal Fgf20a and the later blastemal Fgf3/10a could be responsible for these respective processes. We demonstrated by gain-of-function analyses that Fgf20a induces the expression of distal blastema marker junbl, and that Fgf3 promotes blastema cell proliferation. Our study highlights that Fgfs in the wound epidermis and blastema have distinct functions to regulate fin regeneration cooperatively.
Asunto(s)
Factor 3 de Crecimiento de Fibroblastos/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Proliferación Celular/genética , Proliferación Celular/fisiología , Factor 3 de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM). Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3-BMP signaling axis is a major advance toward understanding how these tissue layers interact during axis extension with important implications in human disease.
Asunto(s)
Proteína Morfogenética Ósea 4/genética , Proteínas Portadoras/genética , Factor 3 de Crecimiento de Fibroblastos/genética , Cresta Neural/metabolismo , Defectos del Tubo Neural/genética , Animales , Tipificación del Cuerpo/genética , Proteína Morfogenética Ósea 4/metabolismo , Proteínas Portadoras/metabolismo , Muerte Celular/genética , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Mesodermo/crecimiento & desarrollo , Mesodermo/metabolismo , Ratones , Cresta Neural/crecimiento & desarrollo , Tubo Neural/crecimiento & desarrollo , Tubo Neural/metabolismo , Defectos del Tubo Neural/metabolismo , Defectos del Tubo Neural/patología , Transducción de Señal/genética , Somitos/crecimiento & desarrollo , Somitos/metabolismoRESUMEN
The mammalian striatum controls sensorimotor and psychoaffective functions through coordinated activities of its two striatonigral and striatopallidal output pathways. Here we show that retinoic acid receptor ß (RARß) controls development of a subpopulation of GABAergic, Gad65-positive striatonigral projection neurons. In Rarb(-/-) knock-out mice, concomitant reduction of Gad65, dopamine receptor D1 (Drd1), and substance P expression at different phases of prenatal development was associated with reduced number of Drd1-positive cells at birth, in contrast to normal numbers of striatopallidal projection neurons expressing dopamine receptor D2. Fate mapping using BrdU pulse-chase experiments revealed that such deficits may originate from compromised proliferation of late-born striosomal neurons and lead to decreased number of Drd1-positive cells retaining BrdU in postnatal day (P) 0 Rarb(-/-) striatum. Reduced expression of Fgf3 in the subventricular zone of the lateral ganglionic eminence (LGE) at embryonic day 13.5 may underlie such deficits by inducing premature differentiation of neuronal progenitors, as illustrated by reduced expression of the proneural gene Ascl1 (Mash1) and increased expression of Meis1, a marker of postmitotic LGE neurons. In agreement with a critical role of FGF3 in this control, reduced number of Ascl1-expressing neural progenitors, and a concomitant increase of Meis1-expressing cells, were observed in primary cell cultures of Rarb(-/-) LGE. This defect was normalized by addition of fibroblast growth factor (FGF). Such data point to role of Meis1 in striatal development, also supported by reduced neuronal differentiation in the LGE of Meis1(-/-) embryos. Our data unveil a novel mechanism of development of striatonigral projection neurons involving retinoic acid and FGF, two signals required for positioning the boundaries of Meis1-expressing cells.
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Cuerpo Estriado/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Proteínas de Homeodominio/fisiología , Proteínas de Neoplasias/fisiología , Neuronas/fisiología , Receptores de Ácido Retinoico/fisiología , Sustancia Negra/fisiología , Animales , Antimetabolitos/farmacología , Bromodesoxiuridina/farmacología , Cuerpo Estriado/citología , Cuerpo Estriado/embriología , Femenino , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Glutamato Descarboxilasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Neurogénesis/genética , Neurogénesis/fisiología , Embarazo , Cultivo Primario de Células , Receptores de Dopamina D1/metabolismo , Sustancia Negra/citología , Sustancia Negra/embriologíaRESUMEN
Here, we developed an isogenic cell model of "stemness" to facilitate protein biomarker discovery in breast cancer. For this purpose, we used knowledge gained previously from the study of the mouse mammary tumor virus (MMTV). MMTV initiates mammary tumorigenesis in mice by promoter insertion adjacent to two main integration sites, namely Int-1 (Wnt1) and Int-2 (Fgf3), which ultimately activates Wnt/ß-catenin signaling, driving the propagation of mammary cancer stem cells (CSCs). Thus, to develop a humanized model of MMTV signaling, we over-expressed WNT1 and FGF3 in MCF7 cells, an ER(+) human breast cancer cell line. We then validated that MCF7 cells over-expressing both WNT1 and FGF3 show a 3.5-fold increase in mammosphere formation, and that conditioned media from these cells is also sufficient to promote stem cell activity in untransfected parental MCF7 and T47D cells, as WNT1 and FGF3 are secreted factors. Proteomic analysis of this model system revealed the induction of i) EMT markers, ii) mitochondrial proteins, iii) glycolytic enzymes and iv) protein synthesis machinery, consistent with an anabolic CSC phenotype. MitoTracker staining validated the expected WNT1/FGF3-induced increase in mitochondrial mass and activity, which presumably reflects increased mitochondrial biogenesis. Importantly, many of the proteins that were up-regulated by WNT/FGF-signaling in MCF7 cells, were also transcriptionally over-expressed in human breast cancer cells in vivo, based on the bioinformatic analysis of public gene expression datasets of laser-captured patient samples. As such, this isogenic cell model should accelerate the discovery of new biomarkers to predict clinical outcome in breast cancer, facilitating the development of personalized medicine.Finally, we used mitochondrial mass as a surrogate marker for increased mitochondrial biogenesis in untransfected MCF7 cells. As predicted, metabolic fractionation of parental MCF7 cells, via MitoTracker staining, indicated that high mitochondrial mass is a new metabolic biomarker for the enrichment of anabolic CSCs, as functionally assessed by mammosphere-forming activity. This observation has broad implications for understanding the role of mitochondrial biogenesis in the propagation of stem-like cancer cells. Technically, this general metabolic approach could be applied to any cancer type, to identify and target the mitochondrial-rich CSC population.The implications of our work for understanding the role of mitochondrial metabolism in viral oncogenesis driven by random promoter insertions are also discussed, in the context of MMTV and ALV infections.
Asunto(s)
Biomarcadores de Tumor/fisiología , Neoplasias de la Mama/patología , Factor 3 de Crecimiento de Fibroblastos/biosíntesis , Mitocondrias/fisiología , Proteína Wnt1/biosíntesis , Medios de Cultivo Condicionados/farmacología , Femenino , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Humanos , Células MCF-7 , Virus del Tumor Mamario del Ratón/genética , Virus del Tumor Mamario del Ratón/patogenicidad , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/metabolismo , Modelos Biológicos , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/patología , Esferoides Celulares/citología , Células Tumorales Cultivadas , Vía de Señalización Wnt/fisiología , Proteína Wnt1/metabolismoRESUMEN
Accumulating evidence has suggested that fibroblast growth factor 3 (FGF3) is expressed in breast cancer and correlates with the stage and grade of the disease. In the present study, a specific FGF3binding peptide (VLWLKNR, termed FP16) was isolated from a phage display heptapeptide library with FGF3. The peptide FP16 contained four identical (WLKN) amino acids and demonstrated high homology to the peptides of the 188194 (TMRWLKN) site of the highaffinity FGF3 receptor fibroblast growth factor receptor 2. Functional analyses indicated that FP16 mediated significant inhibition of FGF3induced cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing proliferationassociated protein 2G4, suppressing cyclin D1 and proliferating cell nuclear antigen, and inhibited the FGF3induced activation of extracellular signalregulated kinase 1/2 and Akt kinase. Taken together, these results demonstrated that the peptide FP16, acting as an FGF3 antagonist, is a promising therapeutic agent for the treatment of breast cancer.
Asunto(s)
Factor 3 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Péptidos/farmacología , Secuencia de Aminoácidos , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Evaluación Preclínica de Medicamentos , Femenino , Factor 3 de Crecimiento de Fibroblastos/genética , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Biblioteca de Péptidos , Péptidos/síntesis química , Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in the tumor stroma, are important modifiers of tumour progression. In the present study, we observed that azoxymethane and dextran sodium sulfate treatments induced increasingly severe colorectal mucosal inflammation and the intratumoural accumulation of CAFs. Fibroblast growth factor (FGF)-1 and FGF-3 were detected in infiltrating cells, and FGFR4, the specific receptor for FGF-1 and FGF-3, was detected in colon cancer tissues. The phosphorylation of FGFR4 enhanced the production of metalloproteinase (MMP)-7 and mitogen-activated protein kinase kinase (Mek)/extracellular signal-regulated kinase (Erk), which was accompanied by excessive vessel generation and cell proliferation. Moreover, we separated CAFs, pericarcinoma fibroblasts (PFs), and normal fibroblasts (NFs) from human colon tissue specimens to characterize the function of CAFs. We observed that CAFs secrete more FGF-1/-3 than NFs and PFs and promote cancer cell growth and angiogenesis through the activation of FGFR4, which is followed by the activation of Mek/Erk and the modulation of MMP-7 expression. The administration of FGF-1/-3-neutralizing antibodies or the treatment of cells with FGFR4 siRNA or the FGFR4 inhibitor PD173074 markedly suppressed colon cancer cell proliferation and neovascularization. These observations suggest a crucial role for CAFs and FGF signaling in the initiation and progression of colorectal cancer. The inhibition of the FGF signaling pathway may be a useful strategy for the treatment of colon cancer.
Asunto(s)
Colitis Ulcerosa/patología , Neoplasias del Colon/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Metaloproteinasa 7 de la Matriz/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Azoximetano , Línea Celular Tumoral , Colitis Ulcerosa/metabolismo , Neoplasias del Colon/genética , Sulfato de Dextran , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Factor 3 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/metabolismo , Células HCT116 , Células HT29 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Fosforilación , Pirimidinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidoresRESUMEN
Wnts and Fgfs regulate various tissues development in vertebrates. However, how regional Wnt or Fgf activities are established and how they interact in any given developmental event is elusive. Here, we investigated the Wnt-mediated craniofacial cartilage development in zebrafish and found that fgf3 expression in the pharyngeal pouches is differentially reduced along the anteroposterior axis in wnt5b mutants and wntless (wls) morphants, but its expression is normal in wnt9a and wnt11 morphants. Introducing fgf3 mRNAs rescued the cartilage defects in Wnt5b- and Wls-deficient larvae. In wls morphants, endogenous Wls expression is not detectable but maternally deposited Wls is present in eggs, which might account for the lack of axis defects in wls morphants. Secretion of endogenous Wnt5b but not Wnt11 was affected in the pharyngeal tissue of Wls morphants, indicating that Wls is not involved in every Wnt secretion event. Furthermore, cell proliferation but not apoptosis in the developing jaw was affected in Wnt5b- and Wls-deficient embryos. Therefore, Wnt5b requires Wls for its secretion and regulates the proliferation of chondrogenic cells through fine-tuning the expression of fgf3 during jaw cartilage development.
Asunto(s)
Cartílago/crecimiento & desarrollo , Proliferación Celular , Condrogénesis/fisiología , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Apoptosis , Cartílago/metabolismo , Células Cultivadas , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Técnicas para Inmunoenzimas , Hibridación in Situ , Morfolinos/farmacología , Proteína Wnt-5a , Pez Cebra/metabolismoRESUMEN
Tooth organogenesis depends on genetically programmed sequential and reciprocal inductive interactions between the dental epithelium and neural crest-derived mesenchyme. Previous studies showed that the Msx1 and Runx2 transcription factors are required for activation of odontogenic signals, including Bmp4 and Fgf3, in the early tooth mesenchyme to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 acts downstream of Msx1 to activate Fgf3 expression. Recent studies identified Osr2 as a repressor of tooth development and showed that inactivation of Osr2 rescued molar tooth morphogenesis in the Msx1(-/-) mutant mice as well as in mice with neural crest-specific inactivation of Bmp4. Here we show that Runx2 expression is expanded in the tooth bud mesenchyme in Osr2(-/-) mutant mouse embryos and is partially restored in the tooth mesenchyme in Msx1(-/-)Osr2(-/-) mutants in comparison with Msx1(-/-) and wild-type embryos. Whereas mandibular molar development arrested at the bud stage and maxillary molar development arrested at the bud-to-cap transition in Runx2(-/-) mutant mice, both mandibular and maxillary molar tooth germs progressed to the early bell stage, with rescued expression of Msx1 and Bmp4 in the dental papilla as well as expression of Bmp4, p21, and Shh in the primary enamel knot in the Osr2(-/-)Runx2(-/-) compound mutants. In contrast to the Msx1(-/-)Osr2(-/-) compound mutants, which exhibit nearly normal first molar morphogenesis, the Osr2(-/-)Runx2(-/-) compound mutant embryos failed to activate the expression of Fgf3 and Fgf10 in the dental papilla and exhibited significant deficit in cell proliferation in both the dental epithelium and mesenchyme in comparison with the control embryos. These data indicate that Runx2 synergizes with Msx1 to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 controls continued tooth growth and morphogenesis beyond the cap stage through activation of Fgf3 and Fgf10 expression in the dental papilla.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Odontogénesis/fisiología , Diente/embriología , Factores de Transcripción/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Muerte Celular , Proliferación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/deficiencia , Femenino , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Etiquetado Corte-Fin in Situ , Factor de Transcripción MSX1/metabolismo , Ratones , Embarazo , Diente/metabolismoRESUMEN
PURPOSE: Laryngeal squamous cell carcinoma (LSCC) represents one of the most common cancers of the head and neck and the search for molecular markers is required for early diagnosis, prognosis and optimal therapy. The purpose of this study was to investigate the clinical significance of Cyclin D1, FGF3, p16 and p21 protein expression in LSCC and laryngeal dysplasia (LD) and to evaluate the associations between their expression levels and clinicopathological parameters of patients with LSCC. METHODS: Immunohistochemistry was employed to detect and quantify the expression levels of Cyclin D1, FGF3, p16 and p21 in the laryngeal tissues of 48 LSCC patients, 32 patients with LD and 28 subjects with healthy laryngeal mucosa (HLM). RESULTS: Significantly higher percentage of LSCC patients had positive Cyclin D1 expression compared with LD patients and HLM subjects (both p<0.01) and positive FGF3 expression than HLM subjects (p<0.05), while no differences in p16 and p21 positive expression were found among studied groups. The levels of Cyclin D1, FGF3 and p16 expression, as evaluated by immunostaining score, were significantly higher in patients with LSCC compared with LD and HLM groups (all p<0.05). Cyclin D1 proved to be highly sensitive and specific marker in differentiating LSCC from LD (sensitivity 81.2%, specificity 83.9%), while high sensitivity (81.2%) and lower specificity (41.4%) was observed in differentiating from HLM. Cyclin D1 and p21 expression levels were associated with regional lymph node metastases (both p<0.05) and Cyclin D1 expression levels significantly correlated with LSCC lymphatic invasion (x(2)=8.862; df=3; ?=0.031). CONCLUSIONS: Cyclin D1, FGF3 and p16 are overexpressed in patients with LSCC. Cyclin D1 is a highly sensitive marker in differentiating LSCC from LD or HLM. Cyclin D1 and p21 expression levels may be useful as predictive markers of metastases in LSCC.