RESUMO
Pluripotency, differentiation, and X Chromosome inactivation (XCI) are key aspects of embryonic development. However, the underlying relationship and mechanisms among these processes remain unclear. Here, we systematically dissected these features along developmental progression using mouse embryonic stem cells (mESCs) and single-cell RNA sequencing with allelic resolution. We found that mESCs grown in a ground state 2i condition displayed transcriptomic profiles diffused from preimplantation mouse embryonic cells, whereas EpiStem cells closely resembled the post-implantation epiblast. Sex-related gene expression varied greatly across distinct developmental states. We also identified novel markers that were highly enriched in each developmental state. Moreover, we revealed that several novel pathways, including PluriNetWork and Focal Adhesion, were responsible for the delayed progression of female EpiStem cells. Importantly, we "digitalized" XCI progression using allelic expression of active and inactive X Chromosomes and surprisingly found that XCI states exhibited profound variability in each developmental state, including the 2i condition. XCI progression was not tightly synchronized with loss of pluripotency and increase of differentiation at the single-cell level, although these processes were globally correlated. In addition, highly expressed genes, including core pluripotency factors, were in general biallelically expressed. Taken together, our study sheds light on the dynamics of XCI progression and the asynchronicity between pluripotency, differentiation, and XCI.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Inativação do Cromossomo X , Animais , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Pluripotentes/metabolismo , Análise de Célula Única , TranscriptomaRESUMO
Single-cell mRNA sequencing can dissect heterogeneous cell populations as it can identify cell types and cellular states based on their unique transcriptional signatures. We use fluorescence-activated cell sorting (FACS) to isolate individual cultured neurons derived from human-induced pluripotent stem cells (hiPSCs) followed by polyA-based Smart-Seq2 RNA sequencing to analyze the single-cell transcriptional profiles. We provide protocols and guidelines on dissociation, cell selection, and library preparation that can be readily adapted to other cell types or tissue samples.
Assuntos
Neurônios , Análise de Célula Única , Humanos , Análise de Célula Única/métodos , Análise de Sequência de RNA/métodos , Biblioteca Gênica , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Perfilação da Expressão Gênica/métodosRESUMO
Oculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Toward this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of PHOX2A in neuronal progenitors. We demonstrate, using electrophysiology, immunocytochemistry, and RNA sequencing, that in vitro-generated neurons are bona fide oculomotor neurons based on their cellular properties and similarity to their in vivo counterpart in rodent and man. We also show that in vitro-generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro that display a resilience similar to that seen in vivo.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Diferenciação Celular , Neurônios Motores/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Transdução de Sinais , Esclerose Lateral Amiotrófica/patologia , Animais , Sobrevivência Celular , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Neurônios Motores/patologia , Células-Tronco Embrionárias Murinas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
LCM-seq couples laser capture microdissection of cells from frozen tissues with polyA-based RNA sequencing and is applicable to single neurons. The method utilizes off-the-shelf reagents and direct lysis of the cells without RNA purification, making it a simple and relatively cheap method with high reproducibility and sensitivity compared to previous methods. The advantage with LCM-seq is also that tissue sections are kept intact and thus the positional information of each cell is preserved.
Assuntos
Perfilação da Expressão Gênica/métodos , Microdissecção e Captura a Laser/métodos , Poli A/metabolismo , Análise de Sequência de RNA/métodos , Animais , Anticorpos/metabolismo , Separação Celular , Células Cultivadas , DNA Complementar/genética , Biblioteca Gênica , Humanos , Camundongos , Coloração e Rotulagem , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
CRISPR/Cas9-based genome editing offers the possibility to knock out almost any gene of interest in an affordable and simple manner. The most common strategy is the introduction of a frameshift into the open reading frame (ORF) of the target gene which truncates the coding sequence (CDS) and targets the corresponding transcript for degradation by nonsense-mediated mRNA decay (NMD). However, we show that transcripts containing premature termination codons (PTCs) are not always degraded efficiently and can generate C-terminally truncated proteins which might have residual or dominant negative functions. Therefore, we recommend an alternative approach for knocking out genes, which combines CRISPR/Cas9 with gene traps (CRISPR-Trap) and is applicable to â¼50% of all spliced human protein-coding genes and a large subset of lncRNAs. CRISPR-Trap completely prevents the expression of the ORF and avoids expression of C-terminal truncated proteins. We demonstrate the feasibility of CRISPR-Trap by utilizing it to knock out several genes in different human cell lines. Finally, we also show that this approach can be used to efficiently generate gene replacements allowing for modulation of protein levels for otherwise lethal knockouts (KOs). Thus, CRISPR-Trap offers several advantages over conventional KO approaches and allows for generation of clean CRISPR/Cas9-based KOs.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Inativação de Genes/métodos , Células HEK293 , Células HeLa , HumanosRESUMO
Single-cell sequencing has emerged as a revolutionary method that reveals biological processes with unprecedented resolution and scale, and has already greatly impacted biology and medicine. To investigate processes such as alternative splicing, novel exon detection and allele-specific expression (ASE), full-length based single-cell RNA-seq methods are required for broad sequence coverage and single nucleotide polymorphism (SNP) identification. In this review, we revisit recent achievements from studies that used single-cell RNA-seq to advance our understanding of ASE in the context of both autosomal and X-chromosome genes. We also recapitulate useful bioinformatic tools developed to identify haplotype phase.
Assuntos
Alelos , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Animais , Genômica , Humanos , Cromossomo X/genéticaRESUMO
Converting resident glia into functional and subtype-specific neurons in vivo by delivering reprogramming genes directly to the brain provides a step forward toward the possibility of treating brain injuries or diseases. To date, it has been possible to obtain GABAergic and glutamatergic neurons via in vivo conversion, but the precise phenotype of these cells has not yet been analyzed in detail. Here, we show that neurons reprogrammed using Ascl1, Lmx1a, and Nurr1 functionally mature and integrate into existing brain circuitry and that the majority of the reprogrammed neurons have properties of fast-spiking, parvalbumin-containing interneurons. When testing different combinations of genes for neural conversion with a focus on pro-neural genes and dopamine fate determinants, we found that functional neurons can be generated using different gene combinations and in different brain regions and that most of the reprogrammed neurons become interneurons, independently of the combination of reprogramming factors used.
Assuntos
Potenciais de Ação , Reprogramação Celular , Interneurônios/metabolismo , Neuroglia/metabolismo , Parvalbuminas/metabolismo , Animais , Diferenciação Celular , Denervação , Dopamina/metabolismo , Regulação da Expressão Gênica , Camundongos , Neostriado/citologia , Fenótipo , Fatores de TempoRESUMO
The fatal disease amyotrophic lateral sclerosis (ALS) is characterized by the loss of somatic motor neurons leading to muscle wasting and paralysis. However, motor neurons in the oculomotor nucleus, controlling eye movement, are for unknown reasons spared. We found that insulin-like growth factor 2 (IGF-2) was maintained in oculomotor neurons in ALS and thus could play a role in oculomotor resistance in this disease. We also showed that IGF-1 receptor (IGF-1R), which mediates survival pathways upon IGF binding, was highly expressed in oculomotor neurons and on extraocular muscle endplate. The addition of IGF-2 induced Akt phosphorylation, glycogen synthase kinase-3ß phosphorylation and ß-catenin levels while protecting ALS patient motor neurons. IGF-2 also rescued motor neurons derived from spinal muscular atrophy (SMA) patients from degeneration. Finally, AAV9::IGF-2 delivery to muscles of SOD1(G93A) ALS mice extended life-span by 10%, while preserving motor neurons and inducing motor axon regeneration. Thus, our studies demonstrate that oculomotor-specific expression can be utilized to identify candidates that protect vulnerable motor neurons from degeneration.