RESUMEN
Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.
Asunto(s)
Síndrome de Down/etiología , Células Madre Pluripotentes Inducidas/fisiología , Diferenciación Celular/fisiología , Síndrome de Down/genética , Síndrome de Down/patología , Femenino , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Neuritas/patología , Neuritas/fisiología , Neurogénesis , Neuronas/patología , Neuronas/fisiología , TranscriptomaRESUMEN
Time series of single-cell transcriptome measurements can reveal dynamic features of cell differentiation pathways. From measurements of whole frog embryos spanning zygotic genome activation through early organogenesis, we derived a detailed catalog of cell states in vertebrate development and a map of differentiation across all lineages over time. The inferred map recapitulates most if not all developmental relationships and associates new regulators and marker genes with each cell state. We find that many embryonic cell states appear earlier than previously appreciated. We also assess conflicting models of neural crest development. Incorporating a matched time series of zebrafish development from a companion paper, we reveal conserved and divergent features of vertebrate early developmental gene expression programs.
Asunto(s)
Diferenciación Celular/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Xenopus/embriología , Xenopus/genética , Animales , Perfilación de la Expresión Génica , Variación Genética , Cresta Neural/citología , Cresta Neural/embriología , Neurogénesis/genética , Células Madre Pluripotentes/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Factores de Transcripción , Transcriptoma , Pez Cebra/embriología , Pez Cebra/genética , CigotoRESUMEN
High-throughput mapping of cellular differentiation hierarchies from single-cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single-cell RNA sequencing to >92,000 cells from zebrafish embryos during the first day of development. Using a graph-based approach, we mapped a cell-state landscape that describes axis patterning, germ layer formation, and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon-based barcoding approach (TracerSeq) for reconstructing single-cell lineage histories. Clonally related cells were often restricted by the state landscape, including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in embryos lacking the chordin gene. We provide web-based resources for further analysis of the single-cell data.
Asunto(s)
Evolución Clonal/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Expresión Génica , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodosRESUMEN
Despite their abundance, the molecular functions of long non-coding RNAs in mammalian nervous systems remain poorly understood. Here we show that the long non-coding RNA, NEAT1, directly modulates neuronal excitability and is associated with pathological seizure states. Specifically, NEAT1 is dynamically regulated by neuronal activity in vitro and in vivo, binds epilepsy-associated potassium channel-interacting proteins including KCNAB2 and KCNIP1, and induces a neuronal hyper-potentiation phenotype in iPSC-derived human cortical neurons following antisense oligonucleotide knockdown. Next generation sequencing reveals a strong association of NEAT1 with increased ion channel gene expression upon activation of iPSC-derived neurons following NEAT1 knockdown. Furthermore, we show that while NEAT1 is acutely down-regulated in response to neuronal activity, repeated stimulation results in NEAT1 becoming chronically unresponsive in independent in vivo rat model systems relevant to temporal lobe epilepsy. We extended previous studies showing increased NEAT1 expression in resected cortical tissue from high spiking regions of patients suffering from intractable seizures. Our results indicate a role for NEAT1 in modulating human neuronal activity and suggest a novel mechanistic link between an activity-dependent long non-coding RNA and epilepsy.
Asunto(s)
Encéfalo/fisiología , Excitabilidad Cortical , Neuronas/fisiología , ARN Largo no Codificante/metabolismo , Convulsiones/patología , Animales , Células Cultivadas , Humanos , Proteínas de Interacción con los Canales Kv/metabolismo , Células Madre Pluripotentes/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , Unión Proteica , Ratas , Canales de Potasio de la Superfamilia ShakerRESUMEN
Only relatively recently has it become clear that mammalian genomes encode tens of thousands of long non-coding RNAs (lncRNAs). A striking 40% of these are expressed specifically in the brain, where they show precisely regulated temporal and spatial expression patterns. This begs the question, what is the functional role of these many lncRNA transcripts in the brain? Here we canvass a growing number of mechanistic studies that have elucidated central roles for lncRNAs in the regulation of nervous system development and function. We also survey studies indicating that neurological and psychiatric disorders may ensue when these mechanisms break down. Finally, we synthesize these insights with evidence from comparative genomics to argue that lncRNAs may have played important roles in brain evolution, by virtue of their abundant sequence innovation in mammals and plausible mechanistic connections to the adaptive processes that occurred recently in the primate and human lineages.
Asunto(s)
Evolución Biológica , Enfermedades del Sistema Nervioso/genética , Sistema Nervioso , Plasticidad Neuronal/fisiología , ARN no Traducido/fisiología , Animales , Humanos , Mamíferos , Sistema Nervioso/anatomía & histología , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Enfermedades del Sistema Nervioso/patologíaRESUMEN
Down syndrome (DS) is a complex developmental disorder with diverse pathologies that affect multiple tissues and organ systems. Clear mechanistic description of how trisomy of chromosome 21 gives rise to most DS pathologies is currently lacking and is limited to a few examples of dosage-sensitive trisomic genes with large phenotypic effects. The recent advent of cellular reprogramming technology offers a promising way forward, by allowing derivation of patient-derived human cell types in vitro. We present general strategies that integrate genomics technologies and induced pluripotent stem cells to identify molecular networks driving different aspects of DS pathogenesis and describe experimental approaches to validate the causal requirement of candidate network defects for particular cellular phenotypes. This overall approach should be applicable to many poorly understood complex human genetic diseases, whose pathogenic mechanisms might involve the combined effects of many genes.