Expression analysis of mouse embryonic stem cell (mESC) derived neuronal cell-types
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE27655 | GEO | 2011/03/04
SECONDARY ACCESSION(S): PRJNA138497
REPOSITORIES: GEO
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Project description:The generation of specific types of neurons from stem cells offers important opportunities in regenerative medicine. However, future applications and proper verification of cell identities will require stringent ways to generate homogenous neuronal cultures. Here we show that under permissive culturing conditions individual transcription factors can induce a desired neuronal lineage from virtually all expressing cells by a mechanism resembling developmental binary cell fate switching. Such efficient selection of cell fate resulted in remarkable cellular enrichment that enabled global gene expression validation of generated neurons and identification of novel features in the studied cell lineages. Several sources of stem cells have a limited competence to differentiate into e.g. dopamine neurons. However, we show that the combination of factors that normally promote either regional or dedicated neuronal specification can overcome limitations in cellular competence and promote efficient reprogramming also in more remote neural contexts, including human neural progenitor cells. We used microarray analysis to verify the identity of several mESC derived neuronal cell types. By genome-wide gene expression comparisons we gained novel insights into molecular properties of several clinical relevant neuronal cell types. Total RNA was extracted from wild-type and transcription-factor induced mESC derived post-mitotic neurons and hybridized on Affymetrix arrays. In total triplicates of 9 different samples were analyzed. Wild-type samples served as control samples.
2011-03-04 | E-GEOD-27655 | biostudies-arrayexpress
Project description:Cell fate specification relies on the action of critical transcription factors that become available at distinct stages of embryonic development. One such factor is NeuroD1, which is essential for eliciting the neuronal development program and possesses the ability to reprogram other cell types into neurons. Given this capacity, it is important to understand its targets and the mechanism underlying neuronal specification. Here, we show that NeuroD1 directly binds regulatory elements of neuronal genes that are developmentally silenced by epigenetic mechanisms. This targeting is sufficient to initiate events that confer transcriptional competence, including reprogramming of transcription factor landscape, conversion of heterochromatin to euchromatin and increased chromatin accessibility, indicating potential pioneer factor ability of NeuroD1. The transcriptional induction of neuronal fate genes is maintained via epigenetic memory despite a transient NeuroD1 induction during neurogenesis. Our study not only reveals the NeuroD1-dependent gene regulatory program driving neurogenesis but also increases our understanding of how cell-fate specification during development involves a concerted action of transcription factors and epigenetic mechanisms. 1. Ectopic NeuroD1 was induced for 48 hours (+Dox) in ES cells for checking initiation of neuronal transcriptional program in comparison to uninduced condition (-Dox) 2. ChIP-seq was performed after 24 hours of NeuroD1 induction in ES cells.
2015-11-02 | E-GEOD-65072 | biostudies-arrayexpress
Project description:This experiment is part of the FunGenES project (FunGenES - "Functional Genomics in Embryonic Stem Cells" partially funded by the 6th Framework Programme of the European Union, http://www.fungenes.org). The experiment was conducted at the "Instituto de Medicina Molecular,Faculdade de Medicina de Lisboa", Portugal. Goal of the experiment is the in vitro generation and characterisation of neurons from embryonic stem (ES) cells. This is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages try to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors (NPs) to their terminal differentiation into neurons and glial cells, is required. In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer system [1]. We established improved conditions to routinely produce highly homogeneous cultures of NPs, which display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. We show that the transition to neuronal production is linked to the organization of NPs into neural tube-like rosettes, where these cells divide and give rise to neurons. In order to characterize the global gene activity correlated with each particular stage of neural differentiation, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five synexpression groups that correlate with successive stages in the path from ES cells to neurons. This resulted in the identification of unique gene signatures for each cell stage, predicting in addition some of the molecular pathways associated with the transitions between these stages. Overall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons.
2009-07-23 | E-TABM-717 | biostudies-arrayexpress
Project description:Ectopic expression of defined transcription factors can force direct cell fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory towards distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 (AS) encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. Intriguingly, during this transient state key signaling components relevant for neural induction and neural stem cell maintenance are regulated and functionally contribute to iN reprogramming and maturation. Thus, AS-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates.
2018-06-05 | GSE113036 | GEO
Project description:Cell fate specification relies on the action of critical transcription factors that become available at distinct stages of embryonic development. One such factor is NeuroD1, which is essential for eliciting the neuronal development program and possesses the ability to reprogram other cell types into neurons. Given this capacity, it is important to understand its targets and the mechanism underlying neuronal specification. Here, we show that NeuroD1 directly binds regulatory elements of neuronal genes that are developmentally silenced by epigenetic mechanisms. This targeting is sufficient to initiate events that confer transcriptional competence, including reprogramming of transcription factor landscape, conversion of heterochromatin to euchromatin and increased chromatin accessibility, indicating potential pioneer factor ability of NeuroD1. The transcriptional induction of neuronal fate genes is maintained via epigenetic memory despite a transient NeuroD1 induction during neurogenesis. Our study not only reveals the NeuroD1-dependent gene regulatory program driving neurogenesis but also increases our understanding of how cell-fate specification during development involves a concerted action of transcription factors and epigenetic mechanisms.
2015-11-02 | GSE65072 | GEO
Project description:The proper balance of excitatory and inhibitory neurons is crucial to normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors, Ascl1 and Ptf1a, are essential for generating the correct number and sub-type of neurons in multiple regions of the nervous system. M-BM- In the dorsal spinal cord, Ascl1 and Ptf1a have contrasting functions in specifying inhibitory versus excitatory neurons. To understand how Ascl1 and Ptf1a function in these processes, we identified their direct transcriptional targets genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. We show that Ascl1 and Ptf1a regulate the specification of excitatory and inhibitory neurons in the dorsal spinal cord through direct regulation of distinct homeodomain transcription factors known for their function in neuronal sub-type specification. Besides their roles in regulating these homeodomain factors, Ascl1 and Ptf1a each function differently during neuronal development with Ascl1 directly regulating genes with roles in several steps of the neurogenic program including, Notch signaling, neuronal differentiation, axon guidance, and synapse formation. In contrast, Ptf1a directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, Ptf1a represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Examination of the Ascl1 and Ptf1a bound sequences shows they are enriched for a common E-Box with a GC core and with additional motifs used by Sox, Rfx, Pou, and Homeodomain factors. Ptf1a bound sequences are uniquely enriched in an E-Box with a GA/TC core and in the binding motif for its co-factor Rbpj, providing two keys to specificity of Ptf1a binding. The direct transcriptional targets identified for Ascl1 and Ptf1a provide a molecular understanding for how they function in neuronal development, particularly as key regulators of homeodomain transcription factors required for neuronal sub-type specification. Examination of gene expression in Ascl1 and Ptf1a lineage cells in the developing neural tube.
2014-06-13 | E-GEOD-55831 | biostudies-arrayexpress
Project description:The proper balance of excitatory and inhibitory neurons is crucial to normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors, Ascl1 and Ptf1a, are essential for generating the correct number and sub-type of neurons in multiple regions of the nervous system. M-BM- In the dorsal spinal cord, Ascl1 and Ptf1a have contrasting functions in specifying inhibitory versus excitatory neurons. To understand how Ascl1 and Ptf1a function in these processes, we identified their direct transcriptional targets genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. We show that Ascl1 and Ptf1a regulate the specification of excitatory and inhibitory neurons in the dorsal spinal cord through direct regulation of distinct homeodomain transcription factors known for their function in neuronal sub-type specification. Besides their roles in regulating these homeodomain factors, Ascl1 and Ptf1a each function differently during neuronal development with Ascl1 directly regulating genes with roles in several steps of the neurogenic program including, Notch signaling, neuronal differentiation, axon guidance, and synapse formation. In contrast, Ptf1a directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, Ptf1a represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Examination of the Ascl1 and Ptf1a bound sequences shows they are enriched for a common E-Box with a GC core and with additional motifs used by Sox, Rfx, Pou, and Homeodomain factors. Ptf1a bound sequences are uniquely enriched in an E-Box with a GA/TC core and in the binding motif for its co-factor Rbpj, providing two keys to specificity of Ptf1a binding. The direct transcriptional targets identified for Ascl1 and Ptf1a provide a molecular understanding for how they function in neuronal development, particularly as key regulators of homeodomain transcription factors required for neuronal sub-type specification. Examination of Ascl1 and Ptf1a genome-wide binding in developing neural tube.
2014-06-13 | E-GEOD-55840 | biostudies-arrayexpress
Project description:Dopamine (DA) neurons modulate neural circuits and behaviors via dopamine release from expansive, long range axonal projections. The elaborate cytoarchitecture of DA neurons is embedded within complex brain tissues, making it difficult to access the DA neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell type-specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of DA neurons. Our dataset reveals the proteomic architecture underlying axonal transport, dopamine transmission, and axonal metabolism in DA neurons. We find a significant enrichment of proteins encoded by Parkinson’s disease-linked genes in dopaminergic axons, including proteins with previously undescribed axonal localization. Our proteomic datasets comprise a significant resource for axonal and DA neuronal cell biology, while the methodology developed here will enable future studies of other neural cell types.
2021-06-01 | PXD026229 | Pride
Project description:With Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron / hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the VZV mimic poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons.
2023-04-19 | GSE228390 | GEO
Project description:BACKGROUND: The polycomb group protein Ezh2 is an epigenetic repressor of transcription originally found to prevent untimely differentiation of pluripotent embryonic stem cells. We previously demonstrated that Ezh2 is also expressed in multipotent neural stem cells (NSCs). We showed that Ezh2 expression is downregulated during NSC differentiation into astrocytes or neurons. However, high levels of Ezh2 remained present in differentiating oligodendrocytes until myelinating. This study aimed to elucidate the target genes of Ezh2 in NSCs and in premyelinating oligodendrocytes (pOLs). METHODOLOGY/PRINCIPAL FINDINGS: We performed chromatin immunoprecipitation followed by high-throughput sequencing to detect the target genes of Ezh2 in NSCs and pOLs. We found 1532 target genes of Ezh2 in NSCs. During NSC differentiation, the occupancy of these genes by Ezh2 was alleviated. However, when the NSCs differentiated into oligodendrocytes, 393 of these genes remained targets of Ezh2. Analysis of the target genes indicated that the repressive activity of Ezh2 in NSCs concerns genes involved in stem cell maintenance, in cell cycle control and in preventing neural differentiation. Among the genes in pOLs that were still repressed by Ezh2 were most prominently those associated with neuronal and astrocytic committed cell lineages. Suppression of Ezh2 activity in NSCs caused loss of stem cell characteristics, blocked their proliferation and ultimately induced apoptosis. Suppression of Ezh2 activity in pOLs resulted in derangement of the oligodendrocytic phenotype, due to re-expression of neuronal and astrocytic genes, and ultimately in apoptosis. CONCLUSIONS/SIGNIFICANCE: Our data indicate that the epigenetic repressor Ezh2 in NSCs is crucial for proliferative activity and maintenance of neural stemness. During differentiation towards oligodendrocytes, Ezh2 repression continues particularly to suppress other neural fate choices. Ezh2 is completely downregulated during differentiation towards neurons and astrocytes allowing transcription of these differentiation programs. The specific fate choice towards astrocytes or neurons is apparently controlled by epigenetic regulators other than Ezh2. Examination of Ezh2 target sites in 2 different primary cells types
2012-07-30 | E-GEOD-31655 | biostudies-arrayexpress