RESUMO
The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.
Assuntos
Processamento Alternativo , Córtex Cerebral/metabolismo , Bases de Dados de Ácidos Nucleicos , Endotélio Vascular/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Transcriptoma , Animais , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/citologia , Camundongos , Análise de Sequência de RNARESUMO
This protocol describes the generation of a rapidly myelinating central nervous system coculture for the study of complex neuronal-glial interactions in vitro. Postnatal rat retinal ganglion cells (RGCs) purified by immunopanning are promoted to cluster into reaggregates and then allowed to extend dense beds of radial axons for 10-14 d. Subsequently, rodent oligodendrocyte precursor cells are purified by immunopanning, transfected if desired, and seeded on top of the RGC reaggregates. Under the conditions described here, compact myelin can be observed within 6 d.
Assuntos
Técnicas de Cocultura , Bainha de Mielina/fisiologia , Oligodendroglia/fisiologia , Nervo Óptico/citologia , Retina/citologia , Células Ganglionares da Retina/fisiologia , Animais , Antígenos/metabolismo , Axônios/fisiologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Dendritos/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Básica da Mielina/metabolismo , Proteoglicanas/metabolismo , Ratos , Células Ganglionares da Retina/citologia , Proteínas tau/metabolismoRESUMO
In this article, we introduce methods for generating rapidly myelinating cocultures with reaggregates of purified retinal ganglion cells and optic nerve oligodendrocyte precursor cells. This coculture system facilitates the study of complex central nervous system neuronal-glial interactions and myelination. It enables control of the extracellular environment and allows the use of transfected, virally infected, mutant, or knockout neurons and/or glial cell types. It is therefore possible to assess the role of various signaling pathways and genes in myelination and node of Ranvier formation.
Assuntos
Proteína Básica da Mielina/metabolismo , Bainha de Mielina/fisiologia , Oligodendroglia/fisiologia , Retina/citologia , Células Ganglionares da Retina/fisiologia , Animais , Antígenos/metabolismo , Axônios/fisiologia , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Inibidores Enzimáticos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Oligodendroglia/citologia , Proteoglicanas/metabolismo , Células-Tronco/citologia , Proteínas tau/metabolismoRESUMO
Astrocytes constitute a major cell population in the brain with a myriad of essential functions, yet we know remarkably little about the signaling pathways and mechanisms that direct astrocyte maturation. To explore the signals regulating astrocyte development, we prospectively purified and cultured immature postnatal rodent astrocytes. We identified fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) as robust trophic factors for immature astrocytes. We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway. In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100ß immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR). In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed. These insights can aid in the further dissection of the mechanisms and pathways controlling astrocyte biology and development.