RESUMEN
OBJECTIVE: Fibroblast growth factor 2 (FGF2) has been reported to play divergent roles in white adipogenic differentiation, however, whether it regulates thermogenesis of fat tissues remains largely unknown. We therefore aimed to investigate the effect of FGF2 on fat thermogenesis and elucidate the underlying mechanisms. METHODS: FGF2-KO and wild-type (WT) mice were fed with chow diet and high-fat diet (HFD) for 14 weeks. The brown and white fat mass, thermogenic capability, respiratory exchange ratio, and hepatic fat deposition were determined. In vitro experiments were conducted to compare the thermogenic ability of FGF2-KO- with WT-derived brown and white adipocytes. Exogenous FGF2 was supplemented to in vitro-cultured WT brown and ISO-induced beige adipocytes. The FGFR inhibitor, PPARγ agonist, and PGC-1α expression lentivirus were used with the aid of technologies including Co-IP, ChIP, and luciferase reporter assay to elucidate the mechanisms underlying the FGF2 regulation of thermogenesis. RESULTS: FGF2 gene disruption results in increased thermogenic capability in both brown and beige fat, supporting by increased UCP1 expression, enhanced respiratory exchange ratio, and elevated thermogenic potential in response to cold exposure. Thus, the deletion of FGF2 protects mice from high fat-induced adiposity and hepatic steatosis. Mechanistically, in vitro investigations indicated FGF2 acts in autocrine/paracrine fashions. Exogenous FGF2 supplementation inhibits both PGC-1α and PPARγ expression, leading to suppression of UCP1 expression in brown and beige adipocytes. CONCLUSIONS: These findings demonstrate that FGF2 is a novel thermogenic regulator, suggesting a viable potential strategy for using FGF2-selective inhibitors in combat adiposity and associated hepatic steatosis.
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Tejido Adiposo Beige/metabolismo , Tejido Adiposo Pardo/metabolismo , Hígado Graso/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Termogénesis , Adiposidad , Animales , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system (CNS) affecting more than two million people worldwide. In MS, oligodendrocytes and myelin sheaths are destroyed by autoimmune-mediated inflammation, while remyelination is impaired. Recent investigations of post-mortem tissue suggest that Fibroblast growth factor (FGF) signaling may regulate inflammation and myelination in MS. FGF2 expression seems to correlate positively with macrophages/microglia and negatively with myelination; FGF1 was suggested to promote remyelination. In myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE), systemic deletion of FGF2 suggested that FGF2 may promote remyelination. Specific deletion of FGF receptors (FGFRs) in oligodendrocytes in this EAE model resulted in a decrease of lymphocyte and macrophage/microglia infiltration as well as myelin and axon degeneration. These effects were mediated by ERK/Akt phosphorylation, a brain-derived neurotrophic factor, and downregulation of inhibitors of remyelination. In the first part of this review, the most important pharmacotherapeutic principles for MS will be illustrated, and then we will review recent advances made on FGF signaling in MS. Thus, we will suggest application of FGFR inhibitors, which are currently used in Phase II and III cancer trials, as a therapeutic option to reduce inflammation and induce remyelination in EAE and eventually MS.
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Encefalomielitis Autoinmune Experimental/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Microglía/inmunología , Esclerosis Múltiple/genética , Vaina de Mielina/inmunología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Regulación de la Expresión Génica , Humanos , Factores Inmunológicos/uso terapéutico , Ratones Noqueados , Microglía/efectos de los fármacos , Microglía/patología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/patología , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Oligodendroglía/efectos de los fármacos , Oligodendroglía/inmunología , Oligodendroglía/patología , Fragmentos de Péptidos/administración & dosificación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/inmunología , Remielinización/efectos de los fármacos , Remielinización/genética , Remielinización/inmunología , Transducción de SeñalRESUMEN
Cancer-associated fibroblasts (CAFs) are known to promote tumourigenesis through various mechanisms. Fibroblast growth factor (FGF)/FGF receptor (FGFR)-dependent lung cancers have been described. We have developed a mouse model of lung adenocarcinoma that was constructed through the induction of Fgf9 overexpression in type 2 alveolar cells. The expression of Fgf9 in adult lungs resulted in the rapid development of multiple adenocarcinoma-like tumour nodules. Here, we have characterised the contribution of CAFs and the Fgf/Fgfr signalling pathway in maintaining the lung tumours initiated by Fgf9 overexpression. We found that CAF-secreted Fgf2 contributes to tumour cell growth. CAFs overexpressed Tgfb, Mmp7, Fgf9, and Fgf2; synthesised more collagen, and secreted inflammatory cell-recruiting cytokines. CAFs also enhanced the conversion of tumour-associated macrophages (TAMs) to the tumour-supportive M2 phenotype but did not influence angiogenesis. In vivo inhibition of Fgfrs during early lung tumour development resulted in significantly smaller and fewer tumour nodules, whereas inhibition in established lung tumours caused a significant reduction in tumour size and number. Fgfr inhibition also influenced tumour stromal cells, as it significantly abolished TAM recruitment and reduced tumour vascularity. However, the withdrawal of the inhibitor caused a significant recurrence/regrowth of Fgf/Fgfr-independent lung tumours. These recurrent tumours did not possess a higher proliferative or propagative potential. Our results provide evidence that fibroblasts associated with the Fgf9-induced lung adenocarcinoma provide multiple means of support to the tumour. Although the Fgfr blocker significantly suppressed the tumour and its stromal cells, it was not sufficient to completely eliminate the tumour, probably due to the emergence of alternative (resistance/maintenance) mechanism(s). This model represents an excellent tool to further study the complex interactions between CAFs, their related chemokines, and the progression of lung adenocarcinoma; it also provides further evidence to support the need for a combinatorial strategy to treat lung cancer. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Adenocarcinoma del Pulmón/tratamiento farmacológico , Antineoplásicos/farmacología , Benzamidas/farmacología , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 9 de Crecimiento de Fibroblastos/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Adenocarcinoma del Pulmón/enzimología , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Animales , Fibroblastos Asociados al Cáncer/enzimología , Fibroblastos Asociados al Cáncer/patología , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/enzimología , Matriz Extracelular/patología , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 9 de Crecimiento de Fibroblastos/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Comunicación Paracrina , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
FGF2 is an essential growth factor implicated in osteoarthritis (OA), and deletion of full-length FGF2 (Fgf2ALLKO ) leads to murine OA. However, the FGF2 gene encodes both high-molecular-weight (HMW) and low-molecular-weight (LMW) isoforms, and the effects of selectively ablating individual isoforms, as opposed to total FGF2, has not been investigated in the context of OA. We undertook this study to examine whether mice lacking HMW FGF2 (Fgf2HMWKO ) or LMW FGF2 (Fgf2LMWKO ) develop OA and to further characterize the observed OA phenotype in Fgf2ALLKO mice. Fgf2HMWKO mice never developed OA, but 6- and 9-month-old Fgf2LMWKO and Fgf2ALLKO mice displayed signs of OA, including eroded articular cartilage, altered subchondral bone and trabecular architecture, and increased OA marker enzyme levels. Even with mechanical induction of OA, Fgf2HMWKO mice were protected against OA, whereas Fgf2LMWKO and Fgf2ALLKO displayed OA-like changes of the subchondral bone. Before exhibiting OA symptoms, Fgf2LMWKO or Fgf2ALLKO joints displayed differential expression of genes encoding key regulatory proteins, including interleukin-1ß, insulin-like growth factor 1, bone morphogenetic protein 4, hypoxia-inducible factor 1, B-cell lymphoma 2, Bcl2-associated X protein, a disintegrin and metalloproteinase with thrombospondin motifs 5, ETS domain-containing protein, and sex-determining region Y box 9. Moreover, Fgf2LMWKO OA cartilage exhibited increased FGF2, FGF23, and FGFR1 expression, whereas Fgf2HMWKO cartilage had increased levels of FGFR3, which promotes anabolism in cartilage. These results demonstrate that loss of LMW FGF2 results in catabolic activity in joint cartilage, whereas absence of HMW FGF2 with only the presence of LMW FGF2 offers protection from OA.
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Hueso Esponjoso/metabolismo , Cartílago Articular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Osteoartritis/metabolismo , Tibia/metabolismo , Animales , Remodelación Ósea , Hueso Esponjoso/diagnóstico por imagen , Hueso Esponjoso/patología , Cartílago Articular/diagnóstico por imagen , Cartílago Articular/patología , Modelos Animales de Enfermedad , Factor 2 de Crecimiento de Fibroblastos/genética , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Masculino , Ratones de la Cepa 129 , Ratones Noqueados , Peso Molecular , Osteoartritis/genética , Osteoartritis/patología , Osteoartritis/prevención & control , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Tibia/diagnóstico por imagen , Tibia/patología , Factores de Tiempo , Microtomografía por Rayos XRESUMEN
We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 G93A -dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.
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Esclerosis Amiotrófica Lateral/enzimología , Astrocitos/enzimología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Neuronas Motoras/enzimología , Músculo Esquelético/enzimología , Superóxido Dismutasa-1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Astrocitos/patología , Muerte Celular , Células Cultivadas , Modelos Animales de Enfermedad , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Factor 2 de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas Motoras/patología , Mutación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Superóxido Dismutasa-1/genéticaRESUMEN
Previous research has shown that fibroblast growth factor 2 protein (FGF2) can act as an anxiolytic and anti-depressive agent in rodents. Levels of hippocampal FGF2 and FGF2 receptors are decreased in post-mortem brains of individuals with mood disorders. No changes in FGF2 were noted in the post-mortem brains of individuals with mood disorders that were successfully treated with anti-depressant medication prior to death. Mutations in the FGF2 gene in humans have been shown to predict non-responsiveness to the therapeutic effects of selective serotonin reuptake inhibitors (SSRIs). These findings suggest that FGF2 may potentially be a target of and/or required for the therapeutic effects of antidepressant medications. To test this, we employed a rodent model of depressive behaviour, chronic variable stress (CVS) in conjunction with antidepressant treatment (fluoxetine) in wild-type (WT) and FGF2 knockout mice (FGF2KO) and examined depressive and anxiety behaviors. Results showed that fluoxetine reversed the effects of CVS on depressive and anxiety behaviours in wild-type mice only, suggesting that the FGF2 gene is indeed necessary for the therapeutic effects of fluoxetine. Interestingly, CVS decreased hippocampal FGF2 levels and fluoxetine partially reversed this effect. Because FGF2 has been previously shown to modify HPA activity through hippocampal glucocorticoid receptors (GR), we examined levels of glucocorticoid receptors and found a decrease in GR in response to CVS, with a further decrease in FGF2KO. No effect of fluoxetine on GR was observed in either WT or FGF2KO mice. This suggests that further changes in glucocorticoid receptors are not necessary for the anti-depressant effects of fluoxetine in WT mice, although decreased glucocorticoid receptors in response to FGF2 deletion may preclude the therapeutic actions of fluoxetine in FGF2KO. Whether astroglia, astroglial functions, or HPA changes are the downstream target of FGF2-mediated changes induced by fluoxetine remains to be determined, however, the current study reaffirms the potential of FGF2 as a novel therapeutic target in the treatment of depression and anxiety disorders.
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Antidepresivos/uso terapéutico , Trastorno Depresivo/tratamiento farmacológico , Factor 2 de Crecimiento de Fibroblastos/genética , Fluoxetina/uso terapéutico , Animales , Antidepresivos/farmacología , Trastornos de Ansiedad/tratamiento farmacológico , Trastornos de Ansiedad/patología , Conducta Animal/efectos de los fármacos , Trastorno Depresivo/patología , Modelos Animales de Enfermedad , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Fluoxetina/farmacología , Expresión Génica/efectos de los fármacos , Hipocampo/metabolismo , Ratones , Ratones Noqueados , Proyectos Piloto , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismoRESUMEN
In previous studies, we described the presence of fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) in human testis and sperm, which are involved in spermatogenesis and in motility regulation. The aim of the present study was to analyze the role of FGF-2 in the maintenance of sperm physiology using FGF-2 knockout (KO) mice. Our results showed that in wild-type (WT) animals, FGF-2 is expressed in germ cells of the seminiferous epithelium, in epithelial cells of the epididymis, and in the flagellum and acrosomal region of epididymal sperm. In the FGF-2 KO mice, we found alterations in spermatogenesis kinetics, higher numbers of spermatids per testis, and enhanced daily sperm production compared with the WT males. No difference in the percentage of sperm motility was detected, but a significant increase in sperm concentration and in sperm head abnormalities was observed in FGF-2 KO animals. Sperm from KO mice depicted reduced phosphorylation on tyrosine residues (a phenomenon that was associated with sperm capacitation) and increased acrosomal loss after incubation under capacitating conditions. However, the FGF-2 KO males displayed no apparent fertility defects, since their mating with WT females showed no differences in the time to delivery, litter size, and pup weight in comparison with WT males. Overall, our findings suggest that FGF-2 exerts a role in mammalian spermatogenesis and that the lack of FGF-2 leads to dysregulated sperm production and altered sperm morphology and function. FGF-2-deficient mice constitute a model for the study of the complex mechanisms underlying mammalian spermatogenesis.
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Factor 2 de Crecimiento de Fibroblastos/deficiencia , Espermatogénesis , Espermatozoides/fisiología , Animales , Peso Corporal , Epidídimo/metabolismo , Femenino , Fertilidad , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Espermatozoides/ultraestructura , Testículo/metabolismoRESUMEN
BACKGROUND/AIMS: Fibroblast growth factor 2 (FGF2) plays a predominant role during angiogenesis in the adventitia and in atherosclerotic plaque. A dilemma exists, however, as to whether angiogenic stimulation by FGF2 for the prevention and treatment of atherogenesis is feasible. The aim of this study is to investigate the effect of the 18-kDa FGF-2 isoform on atherosclerosis progression in high-fat diet-fed apolipoprotein E knockout (ApoE-/-) mice. METHODS: We established a model of atherosclerosis using ApoE and 18-kDa FGF-2 gene double knockout mice. They were randomly divided into three groups depending on the duration of diet: 8 weeks, 12 weeks and 16 weeks. Then, we studied the morphology and inflammatory factor staining in the atherosclerosis plaque of these mice. RESULTS: Knockout of the 18-kDa FGF-2 isoform did not change the metabolic characteristics of the mice. Compared to the control group, knockout of the 18-kDa FGF-2 isoform significantly attenuated atherogenesis, reduced aortic plaques, reduced macrophage infiltration and suppressed oxidative stress in mice fed with a high fat diet at all-time points. CONCLUSIONS: 18-kDa FGF-2 aggravated the inflammatory reaction of atherosclerosis.
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Aterosclerosis/patología , Factor 2 de Crecimiento de Fibroblastos/genética , Macrófagos/inmunología , Estrés Oxidativo , Animales , Aorta/metabolismo , Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Quimiocina CCL2/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Genotipo , Lípidos/sangre , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peso Molecular , Miocardio/patología , NADPH Oxidasa 4/metabolismo , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Fibroblast growth factor 2 (FGF-2) is an abundant growth factor in the brain and exerts multiple functions on neural cells ranging from cell division, cell fate determination to differentiation. However, many details of the molecular mechanisms underlying the diverse functions of FGF-2 are poorly understood. In a comparative microarray analysis of motor sensory cortex (MSC) tissue of adult knockout (FGF-2(-/-)) and control (FGF-2(+/+)) mice, we found a substantial number of regulated genes, which are implicated in cytoskeletal machinery dynamics. Specifically, we found a prominent downregulation of Arhgef6. Arhgef6 mRNA was significantly reduced in the FGF-2(-/-) cortex, and Arhgef6 protein virtually absent, while RhoA protein levels were massively increased and Cdc42 protein levels were reduced. Since Arhgef6 is localized to dendritic spines, we next analyzed dendritic spines of adult FGF2(-/-) and control mouse cortices. Spine densities were significantly increased, whereas mean length of spines on dendrites of layer V of MSC neurons in adult FGF-2(-/-) mice was significantly decreased as compared to respective controls. Furthermore, neurite length in dissociated cortical cultures from E18 FGF-2(-/-) mice was significantly reduced at DIV7 as compared to wildtype neurons. Despite the fact that altered neuronal morphology and alterations in dendritic spines were observed, FGF-2(-/-) mice behave relatively unsuspicious in several behavioral tasks. However, FGF-2(-/-) mice exhibited decreased thermal pain sensitivity in the hotplate-test.
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Espinas Dendríticas/genética , Regulación hacia Abajo/genética , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Proyección Neuronal/genética , Neuronas/citología , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Adaptación Ocular/genética , Animales , Células Cultivadas , Corteza Cerebral/citología , Embrión de Mamíferos , Factor 2 de Crecimiento de Fibroblastos/genética , Masculino , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuritas/fisiología , Fosfopiruvato Hidratasa/metabolismo , Tiempo de Reacción/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Natación/psicología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The brain functions within a specialized environment tightly controlled by brain barrier mechanisms. Understanding the regulation of barrier formation is important for understanding brain development and may also lead to finding new ways to deliver pharmacotherapies to the brain; access of many potentially promising drugs is severely hindered by these barrier mechanisms. The cellular composition of the neurovascular unit of the blood-brain barrier proper and their effects on regulation of its function are beginning to be understood. One hallmark of the neurovascular unit in the adult is the astroglial foot processes that tightly surround cerebral blood vessels. However their role in barrier formation is still unclear. In this study we examined barrier function in newborn, juvenile and adult mice lacking fibroblast growth factor-2 (FGF-2), which has been shown to result in altered astroglial differentiation during development. We show that during development of FGF-2 deficient mice the astroglial contacts with cerebral blood vessels are delayed compared with wild-type animals. However, this delay did not result in changes to the permeability properties of the blood brain barrier as assessed by exclusion of either small or larger sized molecules at this interface. In addition cerebral vessels were positive for tight-junction proteins and we observed no difference in the ultrastructure of the tight-junctions. The results indicate that the direct contact of astroglia processes to cerebral blood vessels is not necessary for either the formation of the tight-junctions or for basic permeability properties and function of the blood-brain barrier. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1201-1212, 2016.
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Astrocitos/metabolismo , Barrera Hematoencefálica/fisiología , Encéfalo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Animales , Encéfalo/irrigación sanguínea , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Ratones , Ratones Noqueados , PermeabilidadRESUMEN
Genetic variations mediate skeletal responsiveness to mechanical unloading, with individual space travelers exhibiting large variations in the extent of bone loss. We previously identified genomic regions harboring several hundred genes that can modulate the magnitude of skeletal adaptation to mechanical unloading. Here, bioinformatic filters aided in shortlisting 30 genes with bone-related and mechanoregulatory roles. The genes CD44, FGF2, NOD2, and Fas, all associated with ERK signaling, were then functionally tested in hindlimb-unloaded (HLU) knockout (KO) mice. Compared to their respective normally ambulating wildtype (WT) controls, all KO strains, except Fas mice, had lower trabecular bone volume, bone volume fraction, and/or trabecular number. For cortical bone and compared to ambulatory WT mice, CD44(-/-) had impaired properties while FGF2(-/-) showed enhanced indices. NOD2(-/-) and Fas(-/-) did not have a cortical phenotype. In all KO and WT groups, HLU resulted in impaired trabecular and cortical indices, primarily due to trabecular tissue loss and mitigation of cortical bone growth. The difference in trabecular separation between HLU and ambulatory controls was significantly greater in CD44(-/-) and NOD2(-/-) mice than in WT mice. In cortical bone, differences in cortical thickness, total pore volume, and cortical porosity between HLU and controls were aggravated in CD44(-/-) mice. In contrast, deletion of NOD2 and Fas genes mitigated the differences in Po.V between HLU and control mice. Together, we narrowed a previous list of QTL-derived candidate genes from over 300 to 30, and showed that CD44, NOD2, and Fas have distinct functions in regulating changes in trabecular and cortical bone indices during unloading.
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Resorción Ósea/genética , Fémur/metabolismo , Receptores de Hialuranos/genética , Sistema de Señalización de MAP Quinasas , Proteína Adaptadora de Señalización NOD2/genética , Receptor fas/genética , Animales , Densidad Ósea , Resorción Ósea/metabolismo , Resorción Ósea/patología , Biología Computacional , Femenino , Fémur/patología , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Factor 2 de Crecimiento de Fibroblastos/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Suspensión Trasera , Receptores de Hialuranos/metabolismo , Masculino , Mecanotransducción Celular , Ratones , Ratones Noqueados , Proteína Adaptadora de Señalización NOD2/deficiencia , Sitios de Carácter Cuantitativo , Microtomografía por Rayos X , Receptor fas/deficienciaRESUMEN
BACKGROUND: The retinal pigment epithelium (RPE), an important tissue monolayer of retina, sustains visual function and retinal homeostasis. In disease conditions such as Retinitis Pigmentosa (RP) and Age related Macular degeneration (AMD), the integrity and functional capacity of RPE monolayer is compromised. Human embryonic stem cells derived RPE (hESC-RPE) is ideal for cell based therapy because of their ability to morphologically and functionally mimic native fetal and adult RPE. However protocols for optimum culture of hESC-RPE are not well established. AIM: To describe a simplified protocol for differentiating human embryonic stem cells (hESC) into retinal pigment epithelial cells. METHODS: hESC (WA09-DL-11) cell lines were grown with standard stem cell culture protocol. After cell colonies were established, basic fibroblast growth factor (bFGF) was deprived (day 0). hESC colonies expressing pigmentation were characterized for expression of RPE65 and Zonular occludens--1 (ZO-1) with immunocytochemistry on days 0, 36, 42, 56 and 70 and western blot analysis on days 0, 40, 48, 53 and 63. In addition, morphological assessment was conducted on transformed cells longitudinally. RESULTS: Pigmented cells were noted 36 days after deprivation of bFGF from growth media. Immunofluorescence demonstrated progressive up regulation of RPE specific proteins (ZO-1 & RPE 65). Immunofluorescence of ZO-1 (in pixels) was (3.08 ± 0.31) on day 42, (5.33 ± 0.89, p = 0.0001) on day 56 and (4.87 ± 0.57, p = 0.0011) on day 70. Similarly expression of RPE 65 was (2.44 ± 0.31) on day 42, which continued to increase (4.23 ± 0.60, p = 0.0011) on day 56 and (5.59 ± 0.36, p < 0.0001) on day 70. Protein expression patterns using western blot confirmed the trends seen in immunofluorescence. Western blot analysis of ZO-1 expression (in optical density unit) was 272.57 ± 31.75 on day 40, 4212.20 ± 911.31 (p = 0.0004) on day 48, 5182.43 ± 1230.38 (p = 0.030) on day 53 and 5848.76 ± 241.04 (p < 0.0001) on day 63. Protein expression of RPE 65 was 1607.64 ± 247.76 on day 40, 2448.07 ± 152.66 on day 48 and (2341.15 ± 52.84) on day 63. hESC-RPE cells displayed a series of specific morphological changes (cytoplasmic, nuclear pigmentary and cell shape) over the course of time frame. By day 70, cells with hexagonal pattern, dark dense nucleus and uniform cytoplasm were noted in densely pigmented RPE colonies. CONCLUSION: bFGF deprivation leads to successful differentiation of hESC into RPE cells. Longitudinal transformative changes were confirmed with measurement of ZO-1 and RPE 65, specific markers of RPE.
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Diferenciación Celular , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Células Madre Embrionarias Humanas/fisiología , Epitelio Pigmentado de la Retina/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Forma de la Célula , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Ratones , Factores de TiempoRESUMEN
Posttranslational modifications of histone tails in chromatin template can result from environmental experiences such as stress and substance abuse. However, the role of epigenetic modifications as potential predisposing factors in affective behavior is less well established. To address this question, we used our selectively bred lines of high responder (bHR) and low responder (bLR) rats that show profound and stable differences in affective responses, with bLRs being prone to anxiety- and depression-like behavior and bHRs prone to addictive behavior. We first asked whether these phenotypes are associated with basal differences in epigenetic profiles. Our results reveal broad between-group differences in basal levels of trimethylated histone protein H3 at lysine 9 (H3K9me3) in hippocampus (HC), amygdala, and nucleus accumbens. Moreover, levels of association of H3K9me3 at Glucocorticoid Receptor (GR) and Fibroblast growth Factor 2 (FGF2) promoters differ reciprocally between bHRs and bLRs in these regions, consistent with these genes' opposing levels of expression and roles in modulating anxiety behavior. Importantly, this basal epigenetic pattern is modifiable by FGF2, a factor that modulates anxiety behavior. Thus, early-life FGF2, which decreases anxiety, altered the levels of H3K9me3 and its binding at FGF2 and GR promoters of bLRs rendering them more similar to bHRs. Conversely, knockdown of HC FGF2 altered both anxiety behavior and levels of H3K9me3 in bHRs, rendering them more bLR-like. These findings implicate FGF2 as a modifier of epigenetic mechanisms associated with emotional responsiveness, and point to H3K9me3 as a key player in the regulation of affective vulnerability.
Asunto(s)
Afecto/fisiología , Conducta Animal/fisiología , Emociones/fisiología , Epigénesis Genética , Factor 2 de Crecimiento de Fibroblastos/genética , Histonas/química , Histonas/metabolismo , Animales , Ensamble y Desensamble de Cromatina , Metilación de ADN , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Técnicas de Silenciamiento del Gen , Hipocampo/metabolismo , Regiones Promotoras Genéticas , Ratas , Receptores de Glucocorticoides/genéticaRESUMEN
Cytopenias occur frequently in systemic lupus erythematosus, rheumatoid arthritis, Felty's syndrome, and large granular lymphocyte (LGL) leukemia, but the bone marrow microenvironment has not been systematically studied. In LGL leukemia (n = 24), retrospective analysis of bone marrow (BM) histopathology revealed severe fibrosis in 15 of 24 patients (63%) in association with the presence of cytopenias, occurrence of autoimmune diseases, and splenomegaly, but was undetectable in control cases with B cell malignancies (n = 11). Fibrosis severity correlated with T cell LGL cell numbers in the BM, but not in the periphery, suggesting deregulation is limited to the BM microenvironment. To identify fibrosis-initiating populations, primary mesenchymal stromal cultures (MSCs) from patients were characterized and found to display proliferation kinetics and overabundant collagen deposition, but displayed normal telomere lengths and osteoblastogenic, chondrogenic, and adipogenic differentiation potentials. To determine the effect of fibrosis on healthy hematopoietic progenitor cells (HPCs), bioartificial matrixes from rat tail or purified human collagen were found to suppress HPC differentiation and proliferation. The ability of patient MSCs to support healthy HSC proliferation was significantly impaired, but could be rescued with collagenase pretreatment. Clustering analysis confirmed the undifferentiated state of patient MSCs, and pathway analysis revealed an inverse relationship between cell division and profibrotic ontologies associated with reduced basic fibroblast growth factor production, which was confirmed by ELISA. Reconstitution with exogenous basic fibroblast growth factor normalized patient MSC proliferation, collagen deposition, and HPC supportive function, suggesting LGL BM infiltration and secondary accumulation of MSC-derived collagen is responsible for hematopoietic failure in autoimmune-associated cytopenias in LGL leukemia.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/deficiencia , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/patología , Células Madre Mesenquimatosas/metabolismo , Pancitopenia/genética , Anciano , Médula Ósea/patología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Proliferación Celular , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/genética , Fibrosis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Linfocítica Granular Grande/complicaciones , Masculino , Persona de Mediana Edad , Pancitopenia/etiología , Estudios Retrospectivos , Telómero/genética , Telómero/metabolismoRESUMEN
Bone morphogenetic protein 2 (BMP-2) is one of the most potent regulators of osteoblast differentiation and bone formation. R-Smads (Smads 1/5/8) are the major transducers for BMPs receptors and, once activated, they are translocated in the nucleus regulating transcription target genes by interacting with various transcription factors. Runx-2 proteins have been shown to interact through their C-terminal segment with Smads and this interaction is required for in vivo osteogenesis. In particular, recruitment of Smads to intranuclear sites is Runx-2 dependent, and Runx-2 factor may accommodate the dynamic targeting of signal transducer to active transcription sites. Previously, we have shown, by in vitro and in vivo experiments, that BMP-2 up-regulated FGF-2 which is important for the maximal responses of BMP-2 in bone. In this study, we found that endogenous FGF2 is necessary for BMP-2 induced nuclear accumulation and co-localization of Runx-2 and phospho-Smads1/5/8, while Runx/Smads nuclear accumulation and co-localization was reduced in Fgf2-/- osteoblasts. Based on these novel data, we conclude that the impaired nuclear accumulation of Runx-2 in Fgf2-/- osteoblasts reduces R-Smads sub-nuclear targeting with a consequent decreased expression of differentiating markers and impaired bone formation in Fgf2 null mice.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Fluorescencia , Ratones , Osteoblastos/enzimología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Cráneo/citología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
The largest part of the peripheral nervous system is the enteric nervous system (ENS). It consists of an intricate network of several enteric neuronal subclasses with distinct phenotypes and functions within the gut wall. The generation of these enteric phenotypes is dependent upon appropriate neurotrophic support during development. Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor-2 (FGF2) play an important role in the differentiation and function of the ENS. A lack of GDNF or its receptor (Ret) causes intestinal aganglionosis in mice, while fibroblast growth factor receptor signaling antagonist is identified as regulating proteins in the GDNF/Ret signaling in the developing ENS. Primary myenteric plexus cultures and wholemount preparations of wild type (WT) and FGF2-knockout mice were used to analyze distinct enteric subpopulations. Fractal dimension (D) as a measure of self-similarity is an excellent tool to analyze complex geometric shape and was applied to classify the subclasses of enteric neurons concerning their individual morphology. As a consequence of a detailed analysis of subpopulation variations, wholemount preparations were stained for the calcium binding proteins calbindin and calretinin. The fractal analysis showed a reliable consistence of subgroups with different fractal dimensions (D) in each culture investigated. Seven different neuronal subtypes could be differentiated according to a rising D. Within the same D, the neurite length revealed significant differences between wild type and FGF2-knockout cultures, while the subclass distribution was also altered. Depending on the morphological characteristics, the reduced subgroup was supposed to be a secretomotor neuronal type, which could be confirmed by calbindin and calretinin staining of the wholemount preparations. These revealed a reduction up to 40 % of calbindin-positive neurons in the FGF2-knockout mouse. We therefore consider FGF2 playing a more important role in the fine-tuning of the ENS during development as previously assumed.
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Sistema Nervioso Entérico/metabolismo , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Neuronas/metabolismo , Animales , Calbindina 2 , Calbindinas , Muerte Celular , Células Cultivadas , Sistema Nervioso Entérico/crecimiento & desarrollo , Sistema Nervioso Entérico/patología , Femenino , Factor 2 de Crecimiento de Fibroblastos/genética , Fractales , Genotipo , Interpretación de Imagen Asistida por Computador , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Neuritas/metabolismo , Neuritas/patología , Neuronas/clasificación , Neuronas/patología , Fenotipo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteína G de Unión al Calcio S100/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motoneuron loss in brain and spinal cord. Mutations in the superoxide dismutase (SOD) 1 gene account for 10-20% of familial ALS patients. The ALS-mouse model over-expressing a mutant human SOD1 (G93A) gene closely mimics human ALS disease. The cause for the selective death of motoneurons is still unclear, but among several pathomechanisms discussed, loss of neurotrophic factors is one possibility. Basic fibroblast growth factor 2 (FGF-2) plays a prominent role in the motor system. In order to evaluate a role of FGF-2 in ALS pathogenesis, double mouse mutants transgenic for the human SOD1 mutation and lacking the endogenous FGF-2 gene were generated. Both heterozygous and homozygous FGF-2 deficient mutant SOD1 mice showed a significant delay in disease onset and less impaired motor performance in comparison to mutant SOD1 mice with normal FGF-2 levels. Survival of the double mouse mutants was significantly prolonged for two weeks. Motoneuron numbers were significantly higher in the double mutants and astrocytosis was diminished at disease endstage. While one would initially have expected that FGF-2 deficiency deteriorates the phenotype of mutant SOD1 animals, our results revealed a protective effect of FGF-2 reduction. In search of the underlying mechanisms, we could show up-regulation of other neurotrophic factors with proven protective effects in the ALS mouse model, ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) in muscle and spinal cord tissue of double mutant animals.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Modelos Animales de Enfermedad , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Desempeño Psicomotor/fisiología , Superóxido Dismutasa/biosíntesis , Factores de Edad , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Destreza Motora/fisiología , Superóxido Dismutasa-1 , Tasa de Supervivencia/tendenciasRESUMEN
Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson's disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development.
Asunto(s)
Neuronas Dopaminérgicas/fisiología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Sustancia Negra/citología , Área Tegmental Ventral/citología , Factores de Edad , Animales , Animales Recién Nacidos , Tipificación del Cuerpo/genética , Bromodesoxiuridina , Recuento de Células , Muerte Celular/genética , Embrión de Mamíferos , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Proteínas con Homeodominio LIM/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurogénesis/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/genética , Sustancia Negra/embriología , Sustancia Negra/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Área Tegmental Ventral/embriología , Área Tegmental Ventral/crecimiento & desarrolloRESUMEN
Fibroblast growth factor 2 (FGF2) positively modulates osteoblast differentiation and bone formation. However, the mechanism(s) is not fully understood. Because the Wnt canonical pathway is important for bone homeostasis, this study focuses on modulation of Wnt/ß-catenin signaling using Fgf2(-/-) mice (FGF2 all isoforms ablated), both in the absence of endogenous FGF2 and in the presence of exogenous FGF2. This study demonstrates a role of endogenous FGF2 in bone formation through Wnt signaling. Specifically, mRNA expression for the canonical Wnt genes Wnt10b, Lrp6, and ß-catenin was decreased significantly in Fgf2(-/-) bone marrow stromal cells during osteoblast differentiation. In addition, a marked reduction of Wnt10b and ß-catenin protein expression was observed in Fgf2(-/-) mice. Furthermore, Fgf2(-/-) osteoblasts displayed marked reduction of inactive phosphorylated glycogen synthase kinase-3ß, a negative regulator of Wnt/ß-catenin pathway as well as a significant decrease of Dkk2 mRNA, which plays a role in terminal osteoblast differentiation. Addition of exogenous FGF2 promoted ß-catenin nuclear accumulation and further partially rescued decreased mineralization in Fgf2(-/-) bone marrow stromal cell cultures. Collectively, our findings suggest that FGF2 stimulation of osteoblast differentiation and bone formation is mediated in part by modulating the Wnt pathway.
Asunto(s)
Diferenciación Celular , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Ratones , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismo , Factores de Tiempo , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Secreted proteins of the fibroblast growth factor (FGF) family play important roles during development of various organ systems. A detailed knowledge of their temporal and spatial expression profiles, especially of closely related FGF family members, are essential to further identification of specific functions in distinct tissues. In the central nervous system dopaminergic neurons of the substantia nigra and their axonal projections into the striatum progressively degenerate in Parkinson's disease. In contrast, FGF-2 deficient mice display increased numbers of dopaminergic neurons. In this study, we determined the expression profiles of all 22 FGF-ligands and 10 FGF-receptor isoforms, in order to clarify, if FGF-2 deficiency leads to compensatory up-regulation of other FGFs in the nigrostriatal system. Three tissues, ventral mesencephalon (VM), striatum (STR) and as reference tissue spinal cord (SC) of wild-type and FGF-2 deficient mice at four developmental stages E14.5, P0, P28, and adult were comparatively analyzed by quantitative RT-PCR. As no differences between the genotypes were observed, a compensatory up-regulation can be excluded. Moreover, this analysis revealed that the majority of FGF-ligands (18/22) and FGF-receptors (9/10) are expressed during normal development of the nigrostriatal system and identified dynamic changes for some family members. By comparing relative expression level changes to SC reference tissue, general alterations in all 3 tissues, such as increased expression of FGF-1, -2, -22, FgfR-2c, -3c and decreased expression of FGF-13 during postnatal development were identified. Further, specific changes affecting only one tissue, such as increased FGF-16 (STR) or decreased FGF-17 (VM) expression, or two tissues, such as decreased expression of FGF-8 (VM, STR) and FGF-15 (SC, VM) were found. Moreover, 3 developmentally down-regulated FGFs (FGF-8b, FGF-15, FGF-17a) were functionally characterized by plasmid-based over-expression in dissociated E11.5 VM cell cultures, however, such a continuous exposure had no influence on the yield of dopaminergic neurons in vitro.