RESUMO
The cerebellum is crucial for sensorimotor coordination. The cerebellar architecture not only requires proper development but also long-term integrity to ensure accurate functioning. Developmental defects such as impaired neuronal migration or neurodegeneration are thus detrimental to the cerebellum and can result in movement disorders including ataxias. In this study, we identify FBXO41 as a novel CNS-specific F-box protein that localizes to the centrosome and the cytoplasm of neurons and demonstrate that cytoplasmic FBXO41 promotes neuronal migration. Interestingly, deletion of the FBXO41 gene results in a severely ataxic gait in mice, which show delayed neuronal migration of granule neurons in the developing cerebellum in addition to deformities and degeneration of the mature cerebellum. We show that FBXO41 is a critical factor, not only for neuronal migration in the cerebellum, but also for its long-term integrity.
Assuntos
Encéfalo/patologia , Movimento Celular/genética , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/patologia , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia , Animais , Animais Recém-Nascidos , Sobrevivência Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Proteínas F-Box/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Fenótipo , Frações Subcelulares/metabolismoRESUMO
Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.
Assuntos
Movimento Celular/fisiologia , Cerebelo/fisiologia , Eletroporação/métodos , Neurônios/fisiologia , Transfecção/métodos , Animais , Axônios/fisiologia , Cerebelo/citologia , Dendritos/fisiologia , Neurônios/citologia , RatosRESUMO
Cells originating from the germ cell lineage retain the remarkable property under special culture conditions to give rise to cells with embryonic stem cell (ESC) properties, such as the multipotent adult germline stem cells (maGSCs) derived from adult mouse testis. To get an insight into the mechanisms that control pluripotency and differentiation in these cells, we studied how differences observed during in vitro differentiation between ESCs and maGSCs are associated with differences at the level of microRNAs (miRNAs). In this work, we provide for a first time a connection between germ cell origin of maGSCs and their specific miRNA expression profile. We found that maGSCs express higher levels of germ cell markers characteristic for primordial germ cells (PGCs) and spermatogonia compared with ESCs. Retained expression of miR-290 cluster has been previously reported in maGSCs during differentiation and it was associated with higher Oct-4 levels. Here, we show that this property is also shared by another pluripotent cell line originating from the germ line, the embryonic germ cells. In addition, we provide proof that the specific miRNA expression profile of maGSCs has an impact on their differentiation potential. Low levels of miR-302 in maGSCs during the first 10 days of leukaemia inhibitory factor deprivation are shown to be necessary for the maintenance of high levels of early germ cell markers.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , MicroRNAs , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem da Célula , Simulação por Computador , Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Masculino , Camundongos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Root hairs are extensions of root epidermal cells and a model system for directional tip growth of plant cells. A previously uncharacterized Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinase gene (PIP5K3) was identified and found to be expressed in the root cortex, epidermal cells, and root hairs. Recombinant PIP5K3 protein was catalytically active and converted phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Arabidopsis mutant plants homozygous for T-DNA-disrupted PIP5K3 alleles were compromised in root hair formation, a phenotype complemented by expression of wild-type PIP5K3 cDNA under the control of a 1500-bp PIP5K3 promoter fragment. Root hair-specific PIP5K3 overexpression resulted in root hair deformation and loss of cell polarity with increasing accumulation of PIP5K3 transcript. Using reestablishment of root hair formation in T-DNA mutants as a bioassay for physiological functionality of engineered PIP5K3 variants, catalytic activity was found to be essential for physiological function, indicating that PtdIns(4,5)P2 formation is required for root hair development. An N-terminal domain containing membrane occupation and recognition nexus repeats, which is not required for catalytic activity, was found to be essential for the establishment of root hair growth. Fluorescence-tagged PIP5K3 localized to the periphery of the apical region of root hair cells, possibly associating with the plasma membrane and/or exocytotic vesicles. Transient heterologous expression of full-length PIP5K3 in tobacco (Nicotiana tabacum) pollen tubes increased plasma membrane association of a PtdIns(4,5)P2-specific reporter in these tip-growing cells. The data demonstrate that root hair development requires PIP5K3-dependent PtdIns(4,5)P2 production in the apical region of root hair cells.