RESUMEN
We investigated the impact of the human-specific gene CHRFAM7A on the function of α7 nicotinic acetylcholine receptors (α7 nAChRs) in two different types of neurons: human-induced pluripotent stem cell (hiPSC)-derived cortical neurons, and superior cervical ganglion (SCG) neurons, taken from transgenic mice expressing CHRFAM7A. dupα7, the gene product of CHRFAM7A, which lacks a major part of the extracellular N-terminal ligand-binding domain, co-assembles with α7, the gene product of CHRNA7. We assessed the receptor function in hiPSC-derived cortical and SCG neurons with Fura-2 calcium imaging and three different α7-specific ligands: PNU282987, choline, and 4BP-TQS. Given the short-lived open state of α7 receptors, we combined the two orthosteric agonists PNU282987 and choline with the type-2 positive allosteric modulator (PAM II) PNU120596. In line with different cellular models used previously, we demonstrate that CHRFAM7A has a major impact on nicotinic α7 nAChRs by reducing calcium transients in response to all three agonists.
RESUMEN
It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.
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Sustitución de Aminoácidos , Ganglios Basales/metabolismo , Evolución Biológica , Factores de Transcripción Forkhead/metabolismo , Vocalización Animal , Animales , Dendritas/metabolismo , Dopamina/metabolismo , Expresión Génica , Heterocigoto , Humanos , Lenguaje , Depresión Sináptica a Largo Plazo , Ratones , Vías Nerviosas , Plasticidad Neuronal , HablaRESUMEN
Mutations in AUTS2 are associated with autism, intellectual disability, and microcephaly. AUTS2 is expressed in the brain and interacts with polycomb proteins, yet it is still unclear how mutations in AUTS2 lead to neurodevelopmental phenotypes. Here we report that when neuronal differentiation is initiated, there is a shift in expression from a long isoform to a short AUTS2 isoform. Yeast two-hybrid screen identified the splicing factor SF3B1 as an interactor of both isoforms, whereas the polycomb group proteins, PCGF3 and PCGF5, were found to interact exclusively with the long AUTS2 isoform. Reporter assays showed that the first exons of the long AUTS2 isoform function as a transcription repressor, but the part that consist of the short isoform acts as a transcriptional activator, both influenced by the cellular context. The expression levels of PCGF3 influenced the ability of the long AUTS2 isoform to activate or repress transcription. Mouse embryonic stem cells (mESCs) with heterozygote mutations in Auts2 had an increase in cell death during in vitro corticogenesis, which was significantly rescued by overexpressing the human AUTS2 transcripts. mESCs with a truncated AUTS2 protein (missing exons 12-20) showed premature neuronal differentiation, whereas cells overexpressing AUTS2, especially the long transcript, showed increase in expression of pluripotency markers and delayed differentiation. Taken together, our data suggest that the precise expression of AUTS2 isoforms is essential for regulating transcription and the timing of neuronal differentiation.
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Diferenciación Celular , Proteínas del Citoesqueleto , Neuronas/citología , Factores de Transcripción , Animales , Exones , Ratones , Fenotipo , Isoformas de Proteínas/genética , Factores de Transcripción/genéticaRESUMEN
A rare mutation affecting the Forkhead-box protein P2 (FOXP2) transcription factor causes a severe monogenic speech and language disorder. Mice carrying an identical point mutation to that observed in affected patients (Foxp2+/R552H mice) display motor deficits and impaired synaptic plasticity in the striatum. However, the consequences of the mutation on neuronal function, in particular in the cerebral cortex, remain little studied. Foxp2 is expressed in a subset of Layer VI cortical neurons. Here, we used Ntsr1-EGFP mice to identify Foxp2+ neurons in the mouse auditory cortex ex vivo. We studied the functional impact of the R552H mutation on the morphologic and functional properties of Layer VI cortical neurons from Ntsr1-EGFP; Foxp2+/R552H male and female mice. The complexity of apical, but not basal dendrites was significantly lower in Foxp2+/R552H cortico-thalamic neurons than in control Foxp2+/+ neurons. Excitatory synaptic inputs, but not inhibitory synaptic inputs, were decreased in Foxp2+/R552H mice. In response, homeostatic mechanisms would be expected to increase neuronal gain, i.e., the conversion of a synaptic input into a firing output. However, the intrinsic excitability of Foxp2+ cortical neurons was lower in Foxp2+/R552H neurons. A-type and delayed-rectifier (DR) potassium currents, two putative transcriptional targets of Foxp2, were not affected by the mutation. In contrast, GABAB/GIRK signaling, another presumed target of Foxp2, was increased in mutant neurons. Blocking GIRK channels strongly attenuated the difference in intrinsic excitability between wild-type (WT) and Foxp2+/R552H neurons. Our results reveal a novel role for Foxp2 in the control of neuronal input/output homeostasis.SIGNIFICANCE STATEMENT Mutations of the Forkhead-box protein 2 (FOXP2) gene in humans are the first known monogenic cause of a speech and language disorder. The Foxp2 mutation may directly affect neuronal development and function in neocortex, where Foxp2 is expressed. Brain imaging studies in patients with a heterozygous mutation in FOXP2 showed abnormalities in cortical language-related regions relative to the unaffected members of the same family. However, the role of Foxp2 in neocortical neurons is poorly understood. Using mice with a Foxp2 mutation equivalent to that found in patients, we studied functional modifications in auditory cortex neurons ex vivo We found that mutant neurons exhibit alterations of synaptic input and GABAB/GIRK signaling, reflecting a loss of neuronal homeostasis.
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Corteza Cerebral/fisiología , Factores de Transcripción Forkhead/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Neuronas/fisiología , Receptores de GABA-B/fisiología , Proteínas Represoras/genética , Tálamo/fisiología , Animales , Corteza Cerebral/citología , Canales de Potasio de Tipo Rectificador Tardío/fisiología , Espinas Dendríticas/fisiología , Fenómenos Electrofisiológicos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/antagonistas & inhibidores , Antagonistas del GABA/farmacología , Masculino , Ratones , Ratones Transgénicos , Mutación , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Sinapsis/fisiología , Tálamo/citologíaRESUMEN
One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using two-photon in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a "protracted" human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in "real time".
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Células Madre Pluripotentes Inducidas/trasplante , Corteza Motora/embriología , Neurogénesis/fisiología , Células Piramidales/trasplante , Animales , Encéfalo/embriología , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Corteza Motora/citología , Células Piramidales/citología , Trasplante HeterólogoRESUMEN
Genetic disruptions of the forkhead box transcription factor FOXP2 in humans cause an autosomal-dominant speech and language disorder. While FOXP2 expression pattern are highly conserved, its role in specific brain areas for mammalian social behaviors remains largely unknown. Here we studied mice carrying a homozygous cortical Foxp2 deletion. The postnatal development and gross morphological architecture of mutant mice was indistinguishable from wildtype (WT) littermates. Unbiased behavioral profiling of adult mice revealed abnormalities in approach behavior towards conspecifics as well as in the reciprocal responses of WT interaction partners. Furthermore mutant mice showed alterations in acoustical parameters of ultrasonic vocalizations, which also differed in function of the social context. Cell type-specific gene expression profiling of cortical pyramidal neurons revealed aberrant regulation of genes involved in social behavior. In particular Foxp2 mutants showed the downregulation of Mint2 (Apba2), a gene involved in approach behavior in mice and autism spectrum disorder in humans. Taken together these data demonstrate that cortical Foxp2 is required for normal social behaviors in mice.
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Conducta Animal , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Factores de Transcripción Forkhead/deficiencia , Eliminación de Gen , Proteínas Represoras/deficiencia , Conducta Social , Animales , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/patología , Homocigoto , Ratones , Ratones Noqueados , Neuronas/metabolismoRESUMEN
Transcriptome analysis of somatic stem cells and their progeny is fundamental to identify new factors controlling proliferation versus differentiation during tissue formation. Here, we generated a combinatorial, fluorescent reporter mouse line to isolate proliferating neural stem cells, differentiating progenitors and newborn neurons that coexist as intermingled cell populations during brain development. Transcriptome sequencing revealed numerous novel long non-coding (lnc)RNAs and uncharacterized protein-coding transcripts identifying the signature of neurogenic commitment. Importantly, most lncRNAs overlapped neurogenic genes and shared with them a nearly identical expression pattern suggesting that lncRNAs control corticogenesis by tuning the expression of nearby cell fate determinants. We assessed the power of our approach by manipulating lncRNAs and protein-coding transcripts with no function in corticogenesis reported to date. This led to several evident phenotypes in neurogenic commitment and neuronal survival, indicating that our study provides a remarkably high number of uncharacterized transcripts with hitherto unsuspected roles in brain development. Finally, we focussed on one lncRNA, Miat, whose manipulation was found to trigger pleiotropic effects on brain development and aberrant splicing of Wnt7b. Hence, our study suggests that lncRNA-mediated alternative splicing of cell fate determinants controls stem-cell commitment during neurogenesis.
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Encéfalo/embriología , Perfilación de la Expresión Génica/métodos , Células-Madre Neurales/fisiología , ARN Largo no Codificante/genética , Empalme Alternativo , Animales , Encéfalo/citología , Corteza Cerebral/citología , Corteza Cerebral/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Transgénicos , Neurogénesis , Neuronas , Fenotipo , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Wnt/genéticaRESUMEN
The acquisition of language and speech is uniquely human, but how genetic changes might have adapted the nervous system to this capacity is not well understood. Two human-specific amino acid substitutions in the transcription factor forkhead box P2 (FOXP2) are outstanding mechanistic candidates, as they could have been positively selected during human evolution and as FOXP2 is the sole gene to date firmly linked to speech and language development. When these two substitutions are introduced into the endogenous Foxp2 gene of mice (Foxp2(hum)), cortico-basal ganglia circuits are specifically affected. Here we demonstrate marked effects of this humanization of Foxp2 on learning and striatal neuroplasticity. Foxp2(hum/hum) mice learn stimulus-response associations faster than their WT littermates in situations in which declarative (i.e., place-based) and procedural (i.e., response-based) forms of learning could compete during transitions toward proceduralization of action sequences. Striatal districts known to be differently related to these two modes of learning are affected differently in the Foxp2(hum/hum) mice, as judged by measures of dopamine levels, gene expression patterns, and synaptic plasticity, including an NMDA receptor-dependent form of long-term depression. These findings raise the possibility that the humanized Foxp2 phenotype reflects a different tuning of corticostriatal systems involved in declarative and procedural learning, a capacity potentially contributing to adapting the human brain for speech and language acquisition.
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Factores de Transcripción Forkhead/fisiología , Aprendizaje/fisiología , Sustitución de Aminoácidos , Animales , Cuerpo Estriado/fisiología , Dopamina/metabolismo , Femenino , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Humanos , Depresión Sináptica a Largo Plazo , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Transgénicos , Destreza Motora/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Especificidad de la Especie , TranscriptomaRESUMEN
Neocortical development requires tightly controlled spatiotemporal gene expression. However, the mechanisms regulating ribosomal complexes and the timed specificity of neocortical mRNA translation are poorly understood. We show that active mRNA translation complexes (polysomes) contain ribosomal protein subsets that undergo dynamic spatiotemporal rearrangements during mouse neocortical development. Ribosomal protein specificity within polysome complexes is regulated by the arrival of in-growing thalamic axons, which secrete the morphogen Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3). Thalamic WNT3 release during midneurogenesis promotes a change in the levels of Ribosomal protein L7 in polysomes, thereby regulating neocortical translation machinery specificity. Furthermore, we present an RNA sequencing dataset analyzing mRNAs that dynamically associate with polysome complexes as neocortical development progresses, and thus may be regulated spatiotemporally at the level of translation. Thalamic WNT3 regulates neocortical translation of two such mRNAs, Foxp2 and Apc, to promote FOXP2 expression while inhibiting APC expression, thereby driving neocortical neuronal differentiation and suppressing oligodendrocyte maturation, respectively. This mechanism may enable targeted and rapid spatiotemporal control of ribosome composition and selective mRNA translation in complex developing systems like the neocortex. SIGNIFICANCE STATEMENT: The neocortex is a highly complex circuit generating the most evolutionarily advanced complex cognitive and sensorimotor functions. An intricate progression of molecular and cellular steps during neocortical development determines its structure and function. Our goal is to study the steps regulating spatiotemporal specificity of mRNA translation that govern neocortical development. In this work, we show that the timed secretion of Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3) by ingrowing axons from the thalamus regulates the combinatorial composition of ribosomal proteins in developing neocortex, which we term the "neocortical ribosome signature." Thalamic WNT3 further regulates the specificity of mRNA translation and development of neurons and oligodendrocytes in the neocortex. This study advances our overall understanding of WNT signaling and the spatiotemporal regulation of mRNA translation in highly complex developing systems.
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Regulación del Desarrollo de la Expresión Génica , Neocórtex/citología , Neurogénesis/fisiología , Biosíntesis de Proteínas , Ribosomas/metabolismo , Tálamo/metabolismo , Proteína Wnt3/metabolismo , Animales , Axones/metabolismo , Ratones , Neocórtex/metabolismo , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/genéticaRESUMEN
Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP-chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections.
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Encéfalo/embriología , Factores de Transcripción Forkhead/genética , Redes Reguladoras de Genes , Neuritas/metabolismo , Proteínas Represoras/genética , Animales , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Cuerpo Estriado/crecimiento & desarrollo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Cultivo Primario de Células , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In the developing nervous system, constitutive activation of the AKT/mTOR (mammalian target of rapamycin) pathway in myelinating glial cells is associated with hypermyelination of the brain, but is reportedly insufficient to drive myelination by Schwann cells. We have hypothesized that it requires additional mechanisms downstream of NRG1/ErbB signaling to trigger myelination in the peripheral nervous system. Here, we demonstrate that elevated levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) have developmental effects on both oligodendrocytes and Schwann cells. By generating conditional mouse mutants, we found that Pten-deficient Schwann cells are enhanced in number and can sort and myelinate axons with calibers well below 1 microm. Unexpectedly, mutant glial cells also spirally enwrap C-fiber axons within Remak bundles and even collagen fibrils, which lack any membrane surface. Importantly, PIP3-dependent hypermyelination of central axons, which is observed when targeting Pten in oligodendrocytes, can also be induced after tamoxifen-mediated Cre recombination in adult mice. We conclude that it requires distinct PIP3 effector mechanisms to trigger axonal wrapping. That myelin synthesis is not restricted to early development but can occur later in life is relevant to developmental disorders and myelin disease.
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Vaina de Mielina/fisiología , Oligodendroglía/fisiología , Fosfatos de Fosfatidilinositol/metabolismo , Células de Schwann/fisiología , Envejecimiento , Animales , Axones/fisiología , Axones/ultraestructura , Encéfalo/fisiología , Encéfalo/ultraestructura , Recuento de Células , Colágeno/metabolismo , Ratones , Ratones Transgénicos , Vaina de Mielina/ultraestructura , Fibras Nerviosas Mielínicas/fisiología , Fibras Nerviosas Mielínicas/ultraestructura , Neuroglía/fisiología , Neuroglía/ultraestructura , Oligodendroglía/ultraestructura , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Células de Schwann/ultraestructura , Nervio Ciático/fisiología , Nervio Ciático/ultraestructuraRESUMEN
The most well-described example of an inherited speech and language disorder is that observed in the multigenerational KE family, caused by a heterozygous missense mutation in the FOXP2 gene. Affected individuals are characterized by deficits in the learning and production of complex orofacial motor sequences underlying fluent speech and display impaired linguistic processing for both spoken and written language. The FOXP2 transcription factor is highly similar in many vertebrate species, with conserved expression in neural circuits related to sensorimotor integration and motor learning. In this study, we generated mice carrying an identical point mutation to that of the KE family, yielding the equivalent arginine-to-histidine substitution in the Foxp2 DNA-binding domain. Homozygous R552H mice show severe reductions in cerebellar growth and postnatal weight gain but are able to produce complex innate ultrasonic vocalizations. Heterozygous R552H mice are overtly normal in brain structure and development. Crucially, although their baseline motor abilities appear to be identical to wild-type littermates, R552H heterozygotes display significant deficits in species-typical motor-skill learning, accompanied by abnormal synaptic plasticity in striatal and cerebellar neural circuits.
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Factores de Transcripción Forkhead/genética , Aprendizaje/fisiología , Destreza Motora/fisiología , Plasticidad Neuronal/genética , Mutación Puntual , Proteínas Represoras/genética , Trastornos del Habla/genética , Alelos , Animales , Heterocigoto , Humanos , Ratones , Ratones Noqueados , Vocalización Animal/fisiologíaRESUMEN
BACKGROUND: Rare mutations affecting the FOXP2 transcription factor cause a monogenic speech and language disorder. We hypothesized that neural pathways downstream of FOXP2 influence more common phenotypes, such as specific language impairment. METHODS: We performed genomic screening for regions bound by FOXP2 using chromatin immunoprecipitation, which led us to focus on one particular gene that was a strong candidate for involvement in language impairments. We then tested for associations between single-nucleotide polymorphisms (SNPs) in this gene and language deficits in a well-characterized set of 184 families affected with specific language impairment. RESULTS: We found that FOXP2 binds to and dramatically down-regulates CNTNAP2, a gene that encodes a neurexin and is expressed in the developing human cortex. On analyzing CNTNAP2 polymorphisms in children with typical specific language impairment, we detected significant quantitative associations with nonsense-word repetition, a heritable behavioral marker of this disorder (peak association, P=5.0x10(-5) at SNP rs17236239). Intriguingly, this region coincides with one associated with language delays in children with autism. CONCLUSIONS: The FOXP2-CNTNAP2 pathway provides a mechanistic link between clinically distinct syndromes involving disrupted language.
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Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Trastornos del Desarrollo del Lenguaje/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Polimorfismo de Nucleótido Simple , Niño , Inmunoprecipitación de Cromatina , Regulación hacia Abajo , Femenino , Factores de Transcripción Forkhead/metabolismo , Marcadores Genéticos , Estudio de Asociación del Genoma Completo , Haplotipos , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
Erythropoietin (Epo) is upregulated by hypoxia and provides protection against apoptosis of erythroid progenitors in bone marrow and brain neurons. Here we show in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of Epo, fibroblast growth factor 2 and vascular endothelial growth factor via hypoxia-inducible factor-1alpha (HIF-1alpha) stabilization. Hypoxic preconditioning protects retinal morphology and function against light-induced apoptosis by interfering with caspase-1 activation, a downstream event in the intracellular death cascade. In contrast, induction of activator protein-1, an early event in the light-stressed retina, is not affected by hypoxia. The Epo receptor required for Epo signaling localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied Epo that crosses the blood retina barrier and prevents apoptosis even when given therapeutically after light insult. Application of Epo may, through the inhibition of apoptosis, be beneficial for the treatment of different forms of retinal disease.
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Proteínas de Unión al ADN/metabolismo , Eritropoyetina/genética , Luz/efectos adversos , Proteínas Nucleares/metabolismo , Degeneración Retiniana/prevención & control , Factores de Transcripción/metabolismo , Animales , Electrorretinografía , Factores de Crecimiento Endotelial/genética , Eritropoyetina/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Hipoxia/fisiopatología , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Linfocinas/genética , Ratones , Ratones Endogámicos BALB C , Degeneración Retiniana/patología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Here we show that conditional deletion of Pten in a subpopulation of adult neural stem cells in the subependymal zone (SEZ) leads to persistently enhanced neural stem cell self-renewal without sign of exhaustion. These Pten null SEZ-born neural stem cells and progenies can follow the endogenous migration, differentiation, and integration pathways and contribute to constitutive neurogenesis in the olfactory bulb. As a result, Pten deleted animals have increased olfactory bulb mass and enhanced olfactory function. Pten null cells in the olfactory bulb can establish normal connections with peripheral olfactory epithelium and help olfactory bulb recovery from acute damage. Following a focal stroke, Pten null progenitors give rise to greater numbers of neuroblasts that migrate to peri-infarct cortex. However, in contrast to the olfactory bulb, no significant long-term survival and integration can be observed, indicating that additional factors are necessary for long-term survival of newly born neurons after stroke. These data suggest that manipulating PTEN-controlled signaling pathways may be a useful step in facilitating endogenous neural stem/progenitor expansion for the treatment of disorders or lesions in regions associated with constitutive neurogenesis.
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Diferenciación Celular/genética , Eliminación de Gen , Neurogénesis/genética , Neuronas/fisiología , Fosfohidrolasa PTEN/genética , Células Madre/fisiología , Factores de Edad , Animales , Células Cultivadas , Masculino , Ratones , Ratones Transgénicos , Neoplasias del Sistema Nervioso/enzimología , Neoplasias del Sistema Nervioso/genética , Neuronas/citología , Neuronas/enzimología , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/fisiología , Transducción de Señal/genética , Olfato/genética , Células Madre/citología , Células Madre/enzimologíaRESUMEN
We previously discovered that mutations of the human FOXP2 gene cause a monogenic communication disorder, primarily characterized by difficulties in learning to make coordinated sequences of articulatory gestures that underlie speech. Affected people have deficits in expressive and receptive linguistic processing and display structural and/or functional abnormalities in cortical and subcortical brain regions. FOXP2 provides a unique window into neural processes involved in speech and language. In particular, its role as a transcription factor gene offers powerful functional genomic routes for dissecting critical neurogenetic mechanisms. Here, we employ chromatin immunoprecipitation coupled with promoter microarrays (ChIP-chip) to successfully identify genomic sites that are directly bound by FOXP2 protein in native chromatin of human neuron-like cells. We focus on a subset of downstream targets identified by this approach, showing that altered FOXP2 levels yield significant changes in expression in our cell-based models and that FOXP2 binds in a specific manner to consensus sites within the relevant promoters. Moreover, we demonstrate significant quantitative differences in target expression in embryonic brains of mutant mice, mediated by specific in vivo Foxp2-chromatin interactions. This work represents the first identification and in vivo verification of neural targets regulated by FOXP2. Our data indicate that FOXP2 has dual functionality, acting to either repress or activate gene expression at occupied promoters. The identified targets suggest roles in modulating synaptic plasticity, neurodevelopment, neurotransmission, and axon guidance and represent novel entry points into in vivo pathways that may be disturbed in speech and language disorders.
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Encéfalo/fisiología , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Trastornos del Lenguaje/genética , Mutación , Regiones Promotoras Genéticas , Trastornos del Habla/genética , Encéfalo/fisiopatología , Línea Celular , Línea Celular Tumoral , Amplificación de Genes , Humanos , Riñón , Neuronas/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , TransfecciónRESUMEN
Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.
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Proliferación Celular , Regulación del Desarrollo de la Expresión Génica/fisiología , Células Madre Multipotentes/fisiología , Neuronas/fisiología , Proteínas Serina-Treonina Quinasas/biosíntesis , Animales , Astrocitos/metabolismo , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Proteína Ácida Fibrilar de la Glía/biosíntesis , Ratones , Ratones Transgénicos , Células Madre Multipotentes/metabolismo , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/biosíntesis , Transactivadores/metabolismoRESUMEN
During mouse embryonic development, the T-box transcription factor Eomes/Tbr2 is expressed in highly dynamic patterns in various progenitor cell types. Those include the undifferentiated cells of the trophectoderm, ingressing nascent mesoderm at the primitive streak, and intermediate progenitor cells of the developing cerebral cortex. We generated an Eomes(GFP)- targeted allele to follow the highly dynamic patterns of Eomes expression and to allow for the identification of novel expression domains. We show that our novel allele recapitulates endogenous gene expression at known sites of expression and confirm our results by anti-Eomes immunofluorescent staining. Using this novel allele we were able to identify previously undocumented domains of Eomes expression within the visceral endoderm and at various locations in the developing and adult mouse brain.
Asunto(s)
Proteínas Fluorescentes Verdes/genética , Proteínas de Dominio T Box/genética , Alelos , Animales , Secuencia de Bases , Cartilla de ADN , Genes Reporteros , Hibridación in Situ , RatonesRESUMEN
Cerebral cortex expansion is a hallmark of mammalian brain evolution; yet, how increased neurogenesis is coordinated with structural and functional development remains largely unclear. The T-box protein TBR2/EOMES is preferentially enriched in intermediate progenitors and supports cortical neurogenesis expansion. Here we show that TBR2 regulates fine-scale spatial and circuit organization of excitatory neurons in addition to enhancing neurogenesis in the mouse cortex. TBR2 removal leads to a significant reduction in neuronal, but not glial, output of individual radial glial progenitors as revealed by mosaic analysis with double markers. Moreover, in the absence of TBR2, clonally related excitatory neurons become more laterally dispersed and their preferential synapse development is impaired. Interestingly, TBR2 directly regulates the expression of Protocadherin 19 (PCDH19), and simultaneous PCDH19 expression rescues neurogenesis and neuronal organization defects caused by TBR2 removal. Together, these results suggest that TBR2 coordinates neurogenesis expansion and precise microcircuit assembly via PCDH19 in the mammalian cortex.