RESUMO
The development of the cerebral cortex depends on numerous parameters, including extracellular cues and microenvironmental factors that also affect gene expression. C-Terminal Binding Proteins (CtBPs) 1 and 2 are transcriptional co-repressors which have been shown to be critically involved in embryonic development. CtBPs are oxygen sensing molecules, and we have previously demonstrated an important role for CtBP1 in integrating oxygen levels and BMP-signaling to influence neural progenitor fate choice. In turn, CtBP2 has been associated with neurodevelopment and neurological disease, and we have shown that CtBP2 acetylation and dimerization, required for proper transcriptional activity, are regulated by microenvironmental oxygen levels. Yet, the putative function of CtBP2 in mammalian cortical development and neurogenesis in vivo is still largely unknown. Here we show that CtBP2 was widely expressed by neural stem and progenitor cells (NSPCs) as well as neurons during cortical development in mice. By using in utero electroporation of siRNA to reduce the levels of CtBP2 mRNA and protein in the developing mouse brain, we found that the NSPC proliferation and migration were largely perturbed, while glial differentiation under these conditions remained unchanged. Our study provides evidence that CtBP2 is required for the maintenance and migration of the NSPCs during mouse cortical development.
Assuntos
Oxirredutases do Álcool/metabolismo , Córtex Cerebral/metabolismo , Proteínas Correpressoras/metabolismo , Neurogênese , Oxirredutases do Álcool/genética , Animais , Córtex Cerebral/embriologia , Proteínas Correpressoras/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuroglia/citologia , Neuroglia/metabolismoRESUMO
Development of the spinal cord requires dynamic and tightly controlled expression of numerous transcription factors. Forkhead Box protein J1 (FoxJ1) is a transcription factor involved in ciliogenesis and is specifically expressed in ependymal cells (ECs) in the adult central nervous system. However, using FoxJ1 fate-mapping mouse lines, we observed that FoxJ1 is also transiently expressed by the progenitors of other neural subtypes during development. Moreover, using a knock-in mouse line, we discovered that FoxJ1 is essential for embryonic progenitors to follow a normal developmental trajectory. FoxJ1 loss perturbed embryonic progenitor proliferation and cell fate determination, and resulted in formation of adult ECs having impaired stem cell potential and an inability to respond to spinal cord injury in both male and female animals. Thus, our study uncovers unexpected developmental functions of FoxJ1 in cell fate determination of subsets of neural cells and suggests that FoxJ1 is critical for maintaining the stem cell potential of ECs into adulthood.
Assuntos
Diferenciação Celular/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/genética , Células-Tronco/citologia , Animais , Epêndima/metabolismo , Feminino , Masculino , Camundongos , Organogênese/fisiologia , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismoRESUMO
Stroke and spinal cord injury (SCI) are among the most frequent causes of central nervous system (CNS) dysfunction, affecting millions of people worldwide each year. The personal and financial costs for affected individuals, their families, and the broader communities are enormous. Although the mammalian CNS exhibits little spontaneous regeneration and self-repair, recent discoveries have revealed that subpopulations of glial cells in the adult forebrain subventricular zone and the spinal cord ependymal zone possess neural stem cell properties. These endogenous neural stem cells react to stroke and SCI by contributing a significant number of new neural cells to formation of the glial scar. These findings have raised hopes that new therapeutic strategies can be designed based on appropriate modulation of endogenous neural stem cell responses to CNS injury. Here, we review the responses of forebrain and spinal cord neural stem cells to stroke and SCI, the role of these responses in restricting injury-induced tissue loss, and the possibility of directing these responses to promote anatomical and functional repair of the CNS.
Assuntos
Isquemia Encefálica/fisiopatologia , Células-Tronco Neurais/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Acidente Vascular Cerebral/fisiopatologia , Animais , Isquemia Encefálica/terapia , Epêndima/fisiopatologia , Humanos , Traumatismos da Medula Espinal/terapia , Nicho de Células-Tronco/fisiologia , Acidente Vascular Cerebral/terapiaRESUMO
The principal neural cell types forming the mature central nervous system (CNS) are now understood to be diverse. This cellular subtype diversity originates to a large extent from the specification of the earlier proliferating progenitor populations during development. Here, we review the processes governing the differentiation of a common neuroepithelial cell progenitor pool into mature neurons, astrocytes, oligodendrocytes, ependymal cells and adult stem cells. We focus on studies performed in mice and involving two distinct CNS structures: the spinal cord and the cerebral cortex. Understanding the origin, specification and developmental regulators of neural cells will ultimately impact comprehension and treatments of neurological disorders and diseases.
Assuntos
Linhagem da Célula , Sistema Nervoso Central/citologia , Organogênese/fisiologia , Células-Tronco/citologia , Animais , CamundongosRESUMO
Ependymal cells in the lateral ventricular wall are considered to be post-mitotic but can give rise to neuroblasts and astrocytes after stroke in adult mice due to insult-induced suppression of Notch signaling. The transcription factor FoxJ1, which has been used to characterize mouse ependymal cells, is also expressed by a subset of astrocytes. Cells expressing FoxJ1, which drives the expression of motile cilia, contribute to early postnatal neurogenesis in mouse olfactory bulb. The distribution and progeny of FoxJ1-expressing cells in rat forebrain are unknown. Here we show using immunohistochemistry that the overall majority of FoxJ1-expressing cells in the lateral ventricular wall of adult rats are ependymal cells with a minor population being astrocytes. To allow for long-term fate mapping of FoxJ1-derived cells, we used the piggyBac system for in vivo gene transfer with electroporation. Using this method, we found that FoxJ1-expressing cells, presumably the astrocytes, give rise to neuroblasts and mature neurons in the olfactory bulb both in intact and stroke-damaged brain of adult rats. No significant contribution of FoxJ1-derived cells to stroke-induced striatal neurogenesis was detected. These data indicate that in the adult rat brain, FoxJ1-expressing cells contribute to the formation of new neurons in the olfactory bulb but are not involved in the cellular repair after stroke.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Elementos de DNA Transponíveis/genética , Eletroporação , Fatores de Transcrição Forkhead/metabolismo , Neurogênese/fisiologia , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Animais , Diferenciação Celular , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Fatores de Transcrição Forkhead/genética , Imuno-Histoquímica , Masculino , Neurogênese/genética , Bulbo Olfatório/citologia , Ratos , Ratos WistarRESUMO
Adult forebrain neurogenesis is dynamically regulated. Multiple families of niche-derived cues have been implicated in this regulation, but the precise roles of key intracellular signaling pathways remain vaguely defined. Here, we show that mammalian target of rapamycin (mTOR) signaling is pivotal in determining proliferation versus quiescence in the adult forebrain neural stem cell (NSC) niche. Within this niche, mTOR complex-1 (mTORC1) activation displays stage specificity, occurring in transiently amplifying (TA) progenitor cells but not in GFAP+ stem cells. Inhibiting mTORC1 depletes the TA progenitor pool in vivo and suppresses epidermal growth factor (EGF)-induced proliferation within neurosphere cultures. Interestingly, mTORC1 inhibition induces a quiescence-like phenotype that is reversible. Likewise, mTORC1 activity and progenitor proliferation decline within the quiescent NSC niche of the aging brain, while EGF administration reactivates the quiescent niche in an mTORC1-dependent manner. These findings establish fundamental links between mTOR signaling, proliferation, and aging-associated quiescence in the adult forebrain NSC niche.
Assuntos
Envelhecimento , Diferenciação Celular/fisiologia , Células-Tronco Neurais/fisiologia , Prosencéfalo/citologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Proteínas do Domínio Duplacortina , Embrião de Mamíferos , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína Glial Fibrilar Ácida/genética , Proteínas de Fluorescência Verde/genética , Humanos , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microdissecção , Proteínas Associadas aos Microtúbulos/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Neuropeptídeos/metabolismo , Fator de Transcrição 2 de Oligodendrócitos , Gravidez , Proteína S6 Ribossômica/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/metabolismo , Serina-Treonina Quinases TOR/genética , Transfecção , Tubulina (Proteína)/metabolismoRESUMO
Neural stem cells have a broad differentiation repertoire during embryonic development and can be reprogrammed to pluripotency comparatively easily. We report that adult neural stem cells can be reprogrammed at very high efficiency to monocytes, a differentiated fate of an unrelated somatic lineage, by ectopic expression of the Ets transcription factor PU.1. The reprogrammed cells display a marker profile and functional characteristics of monocytes and integrate into tissues after transplantation. The failure to reprogram lineage-committed neural cells to monocytes with PU.1 suggests that neural stem cells are uniquely amenable to reprogramming.
Assuntos
Reprogramação Celular , Monócitos/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células-Tronco/metabolismo , Transativadores/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos , Microscopia Confocal , Monócitos/citologia , Neurônios/citologia , Gravidez , Proteínas Proto-Oncogênicas/genética , Células-Tronco/citologia , Fatores de Tempo , Transativadores/genética , Transdução GenéticaRESUMO
Spinal cord ependymal cells display neural stem cell properties in vitro and generate scar-forming astrocytes and remyelinating oligodendrocytes after injury. We report that ependymal cells are functionally heterogeneous and identify a small subpopulation (8% of ependymal cells and 0.1% of all cells in a spinal cord segment), which we denote ependymal A (EpA) cells, that accounts for the in vitro stem cell potential in the adult spinal cord. After spinal cord injury, EpA cells undergo self-renewing cell division as they give rise to differentiated progeny. Single-cell transcriptome analysis revealed a loss of ependymal cell gene expression programs as EpA cells gained signaling entropy and dedifferentiated to a stem-cell-like transcriptional state after an injury. We conclude that EpA cells are highly differentiated cells that can revert to a stem cell state and constitute a therapeutic target for spinal cord repair.
Assuntos
Células-Tronco Neurais , Traumatismos da Medula Espinal , Diferenciação Celular/fisiologia , Humanos , Células-Tronco Neurais/metabolismo , Neuroglia , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismoRESUMO
Targeted ectopic expression of genes in the adult brain is an invaluable approach for studying many biological processes. This can be accomplished by generating transgenic mice or by virally mediated gene transfer, but these methods are costly and labor intensive. We devised a rapid strategy that allows localized in vivo transfection of plasmid DNA within the adult neurogenic niches without detectable brain damage. Injection of plasmid DNA into the ventricular system or directly into the hippocampus of adult mice, followed by application of electrical current via external electrodes, resulted in transfection of neural stem or progenitor cells and mature neurons. We showed that this strategy can be used for both fate mapping and gain- or loss-of-function experiments. Using this approach, we identified an essential role for cadherins in maintaining the integrity of the lateral ventricle wall. Thus, in vivo electroporation provides a new approach to study the adult brain.
Assuntos
Ventrículos Cerebrais/fisiologia , DNA/administração & dosagem , DNA/genética , Eletroporação/métodos , Neurônios/fisiologia , Transfecção/métodos , Animais , CamundongosRESUMO
A fundamental question in stem cell research is whether cultured multipotent adult stem cells represent endogenous multipotent precursor cells. Here we address this question, focusing on SKPs, a cultured adult stem cell from the dermis that generates both neural and mesodermal progeny. We show that SKPs derive from endogenous adult dermal precursors that exhibit properties similar to embryonic neural-crest stem cells. We demonstrate that these endogenous SKPs can first be isolated from skin during embryogenesis and that they persist into adulthood, with a niche in the papillae of hair and whisker follicles. Furthermore, lineage analysis indicates that both hair and whisker follicle dermal papillae contain neural-crest-derived cells, and that SKPs from the whisker pad are of neural-crest origin. We propose that SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood.
Assuntos
Pele/citologia , Células-Tronco/citologia , Adulto , Animais , Western Blotting , Células Cultivadas , Embrião de Galinha , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Cabelo/citologia , Humanos , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.
Assuntos
Linhagem da Célula , Epêndima/patologia , Traumatismos da Medula Espinal/patologia , Células-Tronco/patologia , Animais , Diferenciação Celular , Movimento Celular , Camundongos , Neuroglia/patologia , Neurônios/patologia , Neurônios/fisiologia , Células-Tronco/fisiologiaRESUMO
The ventricular epithelium of the adult forebrain is a heterogeneous cell population that is a source of both quiescent and activated neural stem cells (qNSCs and aNSCs, respectively). We genetically targeted a subset of ventricle-contacting, glial fibrillary acidic protein (GFAP)-expressing cells, to study their involvement in qNSC/aNSC-mediated adult neurogenesis. Ventricle-contacting GFAP+ cells were lineage-traced beginning in early adulthood using adult brain electroporation and produced small numbers of olfactory bulb neuroblasts until at least 21 mo of age. Notably, electroporated GFAP+ neurogenic precursors were distinct from both qNSCs and aNSCs: they did not give rise to neurosphere-forming aNSCs in vivo or after extended passaging in vitro and they were not recruited during niche regeneration. GFAP+ cells with these properties included a FoxJ1+GFAP+ subset, as they were also present in an inducible FoxJ1 transgenic lineage-tracing model. Transiently overexpressing Mash1 increased the neurogenic output of electroporated GFAP+ cells in vivo, identifying them as a potentially recruitable population. We propose that the qNSC/aNSC lineage of the adult forebrain coexists with a distinct, minimally expanding subset of GFAP+ neurogenic precursors.
Assuntos
Ventrículos Cerebrais/metabolismo , Epitélio/metabolismo , Marcação de Genes , Fatores de Crescimento Neural/genética , Células-Tronco Neurais/metabolismo , Prosencéfalo/metabolismo , Adulto , Células-Tronco Adultas/metabolismo , Animais , Biomarcadores , Diferenciação Celular/genética , Imunofluorescência , Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Fatores de Crescimento Neural/metabolismo , Células-Tronco Neurais/citologia , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Nicho de Células-Tronco/genéticaRESUMO
Precursor cells of the embryonic cortex sequentially generate neurons and then glial cells, but the mechanisms regulating this neurogenic-to-gliogenic transition are unclear. Using cortical precursor cultures, which temporally mimic this in vivo differentiation pattern, we demonstrate that cortical neurons synthesize and secrete the neurotrophic cytokine cardiotrophin-1, which activates the gp130-JAK-STAT pathway and is essential for the timed genesis of astrocytes in vitro. Our data indicate that a similar phenomenon also occurs in vivo. In utero electroporation of neurotrophic cytokines in the environment of embryonic cortical precursors causes premature gliogenesis, while acute perturbation of gp130 in cortical precursors delays the normal timed appearance of astrocytes. Moreover, the neonatal cardiotrophin-1-/- cortex contains fewer astrocytes. Together, these results describe a neural feedback mechanism; newly born neurons produce cardiotrophin-1, which instructs multipotent cortical precursors to generate astrocytes, thereby ensuring that gliogenesis does not occur until neurogenesis is largely complete.
Assuntos
Diferenciação Celular/fisiologia , Córtex Cerebral/citologia , Citocinas/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Células-Tronco , Análise de Variância , Animais , Western Blotting/métodos , Contagem de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/embriologia , Fator Neurotrófico Ciliar/farmacologia , Contactinas , Meios de Cultivo Condicionados/farmacologia , Quinase 2 Dependente de Ciclina/metabolismo , Citocinas/deficiência , Citocinas/farmacologia , Interações Medicamentosas , Proteínas ELAV/metabolismo , Embrião de Mamíferos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Imunofluorescência/métodos , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Receptores de Hialuronatos/metabolismo , Interleucina-6/farmacologia , Proteínas de Filamentos Intermediários/metabolismo , Fator Inibidor de Leucemia , Camundongos , Camundongos Transgênicos , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nestina , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteínas de Neurofilamentos/metabolismo , Organogênese , Fosfopiruvato Hidratase/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Fatores de Transcrição STAT/metabolismo , Células-Tronco/efeitos dos fármacos , Fatores de Tempo , Transfecção/métodos , Tubulina (Proteína)/metabolismo , Tirfostinas/farmacologiaRESUMO
The p53 family member p63 is required for nonneural development, but has no known role in the nervous system. Here, we define an essential proapoptotic role for p63 during naturally occurring neuronal death. Sympathetic neurons express full-length TAp63 during the developmental death period, and TAp63 levels increase following NGF withdrawal. Overexpression of TAp63 causes neuronal apoptosis in the presence of NGF, while cultured p63-/- neurons are resistant to apoptosis following NGF withdrawal. TAp63 is also essential in vivo, since embryonic p63-/- mice display a deficit in naturally occurring sympathetic neuron death. While both TAp63 and p53 induce similar apoptotic signaling proteins and require BAX expression and function for their effects, TAp63 induces neuronal death in the absence of p53, but p53 requires coincident p63 expression for its proapoptotic actions. Thus, p63 is essential for developmental neuronal death, likely functioning both on its own, and as an obligate proapoptotic partner for p53.
Assuntos
Apoptose/fisiologia , Fosfoproteínas/fisiologia , Sistema Nervoso Simpático/embriologia , Sistema Nervoso Simpático/crescimento & desenvolvimento , Transativadores/fisiologia , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Morte Celular/fisiologia , Células Cultivadas , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Mutantes , Mitocôndrias/fisiologia , Fator de Crescimento Neural/administração & dosagem , Fator de Crescimento Neural/farmacologia , Neurônios/metabolismo , Neurônios/fisiologia , Fosfoproteínas/deficiência , Fosfoproteínas/metabolismo , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Sistema Nervoso Simpático/citologia , Transativadores/deficiência , Transativadores/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Proteína X Associada a bcl-2/fisiologiaRESUMO
Mammalian neurogenesis is determined by an interplay between intrinsic genetic mechanisms and extrinsic cues such as growth factors. Here we have defined a signaling cascade, a MEK-C/EBP pathway, that is essential for cortical progenitor cells to become postmitotic neurons. Inhibition of MEK or of the C/EBP family of transcription factors inhibits neurogenesis while expression of a C/EBPbeta mutant that is a phosphorylation-mimic at a MEK-Rsk site enhances neurogenesis. C/EBP mediates this positive effect by direct transcriptional activation of neuron-specific genes such as Talpha1 alpha-tubulin. Conversely, inhibition of C/EBP-dependent transcription enhances CNTF-mediated generation of astrocytes from the same progenitor cells. Thus, activation of a MEK-C/EBP pathway enhances neurogenesis and inhibits gliogenesis, thereby providing a mechanism whereby growth factors can selectively bias progenitors to become neurons during development.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Córtex Cerebral/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Substâncias de Crescimento/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Fator Neurotrófico Ciliar/metabolismo , Fator Neurotrófico Ciliar/farmacologia , Feto , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , MAP Quinase Quinase 1 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fator de Transcrição CHOP , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
The intracellular mechanisms that bias mammalian neural precursors to generate neurons versus glial cells are not well understood. We demonstrated previously that the growth factor-regulated mitogen-activated protein kinase kinase (MEK) and its downstream target, the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, are essential for neurogenesis in cultured cortical precursor cells (Ménard et al., 2002). Here, we examined a role for this pathway during cortical cell fate determination in vivo using in utero electroporation of the embryonic cortex. These studies demonstrate that inhibition of the activity of either MEK or the C/EBPs inhibits the genesis of neurons in vivo. Moreover, the MEK pathway mediates phosphorylation of C/EBPbeta in cortical precursors, and expression of a C/EBPbeta construct in which the MEK pathway phosphorylation sites are mutated inhibits neurogenesis. Conversely, expression of a C/EBPbeta construct, in which the same sites are mutated to glutamate and therefore are "constitutively" phosphorylated, enhances neurogenesis in the early embryonic cortex. A subpopulation of precursors in which C/EBP activity is inhibited are maintained as cycling precursors in the ventricular/subventricular zone of the cortex until early in postnatal life, when they have an enhanced propensity to generate astrocytes, presumably in response to gliogenic signals in the neonatal environment. Thus, activation of an MEK-C/EBP pathway in cortical precursors in vivo biases them to become neurons and against becoming astrocytes, thereby acting as a growth factor-regulated switch.
Assuntos
Astrócitos/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular/fisiologia , Córtex Cerebral/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Animais , Astrócitos/citologia , Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Linhagem Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , Humanos , MAP Quinase Quinase 1/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Neurônios/citologia , Neurônios/enzimologia , Fosforilação , Células-Tronco/citologia , Células-Tronco/enzimologiaRESUMO
Neurogenesis requires factors that regulate the decision of dividing progenitors to leave the cell cycle and activate the neuronal differentiation program. It is shown here that the murine runt-related gene Runx1 is expressed in proliferating cells on the basal side of the olfactory epithelium. These include both Mash1+ olfactory receptor neuron (ORN) progenitors and NeuroD+ ORN precursors. Disruption of Runx1 function in vivo does not cause a change in Mash1 expression but leads to a decrease in the number of NeuroD+ neuronal precursors and an increase in differentiated ORNs. These effects result in premature and ectopic ORN differentiation. It is shown further that exogenous Runx1 expression in cultured olfactory neural progenitors causes an expansion of the mitotic cell population. In agreement with these findings, exogenous Runx1 expression also promotes cortical neural progenitor cell proliferation without inhibiting neuronal differentiation. These effects are phenocopied by a chimeric protein containing ETO, the eight twenty one transcriptional repressor, fused to the Runx1 DNA-binding domain, which suggests the involvement of transcription repression mechanisms. Consistent with this possibility, Runx1 represses transcription driven by the promoter of the cell cycle inhibitor p21Cip 1 in cortical progenitors. Together, these findings suggest a previously unrecognized role for Runx1 in coordinating the proliferation and neuronal differentiation of selected populations of neural progenitors.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/fisiologia , Mucosa Olfatória/citologia , Neurônios Receptores Olfatórios/citologia , Proteínas Proto-Oncogênicas/fisiologia , Células-Tronco/citologia , Telencéfalo/citologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Substituição de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core , Inibidor de Quinase Dependente de Ciclina p21 , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Mucosa Olfatória/embriologia , Neurônios Receptores Olfatórios/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/fisiologia , Mutação Puntual , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteína 1 Parceira de Translocação de RUNX1 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Células-Tronco/metabolismo , Telencéfalo/embriologia , Fatores de Transcrição/genéticaRESUMO
Stem cells have a high therapeutic potential for the treatment of spinal cord injury (SCI). We have shown previously that endogenous stem cell potential is confined to ependymal cells in the adult spinal cord which could be targeted for non-invasive SCI therapy. However, ependymal cells are an understudied cell population. Taking advantage of transgenic lines, we characterize the appearance and potential of ependymal cells during development. We show that spinal cord stem cell potential in vitro is contained within these cells by birth. Moreover, juvenile cultures generate more neurospheres and more oligodendrocytes than adult ones. Interestingly, juvenile ependymal cells in vivo contribute to glial scar formation after severe but not mild SCI, due to a more effective sealing of the lesion by other glial cells. This study highlights the importance of the age-dependent potential of stem cells and post-SCI environment in order to utilize ependymal cell's regenerative potential.
Assuntos
Diferenciação Celular , Epêndima/citologia , Células-Tronco Neurais/citologia , Regeneração , Traumatismos da Medula Espinal/patologia , Animais , Autorrenovação Celular , Células Cultivadas , Modelos Animais de Doenças , Genes Reporter , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microglia/imunologia , Microglia/metabolismo , Células-Tronco Neurais/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/terapiaRESUMO
Cultured embryonic cortical progenitor cells will mimic the temporal differentiation pattern observed in vivo, producing neurons first and then glia. Here, we investigated the role of two endogenously produced growth factors, the neurotrophins brain-derived neurotrophic factor and neurotrophin-3 (NT-3), in the early progenitor-to-neuron transition. Cultured cortical progenitors express BDNF and NT-3, as well as their receptors TrkB (tyrosine kinase receptor B) and TrkC. Inhibition of these endogenously expressed neurotrophins using function-blocking antibodies resulted in a marked decrease in the survival of cortical progenitors, accompanied by decreased proliferation and inhibition of neurogenesis. Inhibition of neurotrophin function also suppressed the downstream Trk receptor signaling pathways, PI3-kinase (phosphatidyl inositol-3-kinase) and MEK-ERK (MAP kinase kinase-extracellular signal-regulated kinase), indicating the presence of autocrine-paracrine neurotrophin:Trk receptor signaling in these cells. Moreover, specific inhibition of these two Trk signaling pathways led to distinct biological effects; inhibition of PI3-kinase decreased progenitor cell survival, whereas inhibition of MEK selectively blocked the generation of neurons, with no effects on survival or proliferation. Thus, neurotrophins made by cortical progenitor cells themselves signal through the TrkB and TrkC receptors to mediate cortical progenitor cell survival and neurogenesis via two distinct downstream signaling pathways.
Assuntos
Diferenciação Celular/fisiologia , Córtex Cerebral/metabolismo , Fatores de Crescimento Neural/fisiologia , Proteínas Serina-Treonina Quinases , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Animais , Anticorpos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Inibidores Enzimáticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/farmacologia , Neurotrofina 3/antagonistas & inibidores , Neurotrofina 3/farmacologia , Neurotrofina 3/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptor trkB/metabolismo , Receptor trkC/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
In most postmitotic neurons, expression or activation of proteins that stimulate cell cycle progression or DNA replication results in apoptosis. One potential exception to this generalization is neuroblastoma (NB), a tumor derived from the sympathoadrenal lineage. NBs often express high levels of N-myc, a proto-oncogene that can potently activate key components of the cell cycle machinery. Here, we show that in postmitotic sympathetic neurons, N-myc can induce S-phase entry while protecting neurons from death caused by aberrant cell cycle reentry. Specifically, these experiments demonstrate that expression of N-myc at levels similar to those in NBs caused sympathetic neurons to reenter S-phase, as monitored by 5-bromo-2-deoxyuridine incorporation and expression of cell cycle regulatory proteins, and rescued them from apoptosis induced by withdrawal of their obligate survival factor, nerve growth factor. The N-myc-induced cell cycle entry, but not enhanced survival, was inhibited by coexpression of a constitutively hypophosphorylated form of the retinoblastoma tumor suppressor protein, suggesting that these two effects of N-myc are mediated by separate pathways. In contrast, N-myc did not cause S-phase entry in postmitotic cortical neurons. Thus, N-myc both selectively causes sympathetic neurons to reenter the cell cycle and protects them from apoptosis, potentially contributing to their transformation to NBs.