Gsx2 Regulates Oligodendrocyte Precursor Formation in the Zebrafish Spinal Cord
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE316142 | GEO | 2026/01/12
REPOSITORIES: GEO
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Project description:We show that a synthetic modified messenger RNA (smRNA)-based reprogramming method that leads to the generation of transgene-free OLs has been developed. An smRNA encoding a modified form of OLIG2, a key TF in OL development, in which the serine 147 phosphorylation site is replaced with alanine, OLIG2S147A, is designed to reprogram hiPSCs into OLs. We demonstrate that repeated administration of the smRNA encoding OLIG2 S147A lead to higher and more stable protein expression. Using the single-mutant OLIG2 smRNA morphogen, we establish a 6-day smRNA transfection protocol, and glial induction lead to rapid NG2+ OL progenitor cell (OPC) generation (> 70% purity) from hiPSC-derived neural progenitor cells (NPCs). The smRNA-induced NG2+ OPCs can mature into functional OLs in vitro and promote remyelination in vivo. Proteomic analysis of OLIG2-binding proteins indicates that OLIG2 is bound by the heat shock protein 70 (HSP70) complex. The HSP70 complex is bound more strongly to OLIG2 with the modified phosphorylation site than to wild-type OLIG2.
2023-03-10 | PXD036975 | Pride
Project description:Endogenous oligodendrocyte progenitor cells (OPCs) are a promising target to improve functional recovery after spinal cord injury (SCI) by remyelinating denuded, and therefore vulnerable, axons. Demyelination is the result of a primary insult and secondary injury, leading to conduction blocks and long-term degeneration of the axons, which subsequently can lead to the loss of their neuron. In response to SCI, dormant OPCs can be activated and subsequently start to proliferate and differentiate into mature myelinating oligodendrocytes (OLs). Therefore, researchers strive to control OPC responses, and utilize small molecule screening approaches in order to identify mechanisms of OPC activation, proliferation, migration and differentiation.
2017-09-05 | GSE100821 | GEO
Project description:The differentiation of oligodendroglia from oligodendrocyte precursor cells (OPCs) to complex and extensive myelinating oligodendrocytes (OLs) is a multistep process that involves largescale morphological changes with significant strain on the cytoskeleton. While key chromatin and transcriptional regulators of differentiation have been identified, their target genes responsible for the morphological changes occurring during OL myelination are still largely unknown. Here, we show that the regulator of focal adhesion, Tensin3 (Tns3), is a direct target gene of Olig2, Chd7, and Chd8, transcriptional regulators of OL differentiation. Tns3 is transiently upregulated and localized to cell processes of immature OLs, together with integrin-b1, a key mediator of survival at this transient stage. Constitutive Tns3 loss-of-function leads to reduced viability in mouse and humans, with surviving knockout mice still expressing Tns3 in oligodendroglia. Acute deletion of Tns3 in vivo, either in postnatal neural stem cells (NSCs) or in OPCs, leads to a two-fold reduction in OL numbers. We find that the transient upregulation of Tns3 is required to protect differentiating OPCs and immature OLs from cell death by preventing the upregulation of p53, a key regulator of apoptosis. Altogether, our findings reveal a specific time window during which transcriptional upregulation of Tns3 in immature OLs is required for OL differentiation likely by mediating integrin-b1 survival signaling to the actin cytoskeleton as OL undergo the large morphological changes required for their terminal differentiation.
2022-09-01 | GSE203294 | GEO
Project description:The differentiation of oligodendroglia from oligodendrocyte precursor cells (OPCs) to complex and extensive myelinating oligodendrocytes (OLs) is a multistep process that involves largescale morphological changes with significant strain on the cytoskeleton. While key chromatin and transcriptional regulators of differentiation have been identified, their target genes responsible for the morphological changes occurring during OL myelination are still largely unknown. Here, we show that the regulator of focal adhesion, Tensin3 (Tns3), is a direct target gene of Olig2, Chd7, and Chd8, transcriptional regulators of OL differentiation. Tns3 is transiently upregulated and localized to cell processes of immature OLs, together with integrin-b1, a key mediator of survival at this transient stage. Constitutive Tns3 loss-of-function leads to reduced viability in mouse and humans, with surviving knockout mice still expressing Tns3 in oligodendroglia. Acute deletion of Tns3 in vivo, either in postnatal neural stem cells (NSCs) or in OPCs, leads to a two-fold reduction in OL numbers. We find that the transient upregulation of Tns3 is required to protect differentiating OPCs and immature OLs from cell death by preventing the upregulation of p53, a key regulator of apoptosis. Altogether, our findings reveal a specific time window during which transcriptional upregulation of Tns3 in immature OLs is required for OL differentiation likely by mediating integrin-b1 survival signaling to the actin cytoskeleton as OL undergo the large morphological changes required for their terminal differentiation.
2022-09-01 | GSE203293 | GEO
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2023-03-04 | GSE225580 | GEO
Project description:The pMN domain is a restricted domain in the ventral spinal cords, defined by the expression of olig2 gene. The fate determination of pMN progenitors is highly temporally and spatially regulated, with motor neurons and oligodendrocyte progenitor cells (OPCs) developing sequentially. Insight into the heterogeneity and molecular programs of pMN progenitors is currently lacking. With the zebrafish model, we identified multiple states of neural progenitors using single-cell sequencing: proliferating progenitors, common progenitors for both motor neurons and OPCs, and restricted precursors for either motor neurons or OPCs. We found specific molecular programs for neural progenitor fate transition, and manipulations of representative genes in the motor neuron or OPC lineage confirmed their critical role in cell fate determination. Deciphering progenitor heterogeneity and molecular mechanisms for these transitions will elucidate the formation of complex neuron-glia networks in the central nervous system during development, and understand the basis of neurodevelopmental disorders.
2022-08-20 | GSE179096 | GEO
Project description:A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function Understanding the cell-cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate astrocytes, neurons, and oligodendrocytes. Here we describe the first method for the isolation and purification of developing and mature astrocytes from mouse forebrain. This method takes advantage of the expression of S100β by astrocytes. We used fluorescent activated cell sorting (FACS) to isolate EGFP positive cells from transgenic mice that express EGFP under the control of an S100β promoter. By depletion of astrocytes and oligodendrocytes we obtained purified populations of neurons, while by panning with oligodendrocyte-specific antibodies we obtained purified populations of oligodendrocytes. Using GeneChip Arrays we then created a transcriptome database of the expression levels of over 20,000 genes by gene profiling these three main CNS neural cell types at postnatal ages day 1 to 30. This database provides the first global characterization of the genes expressed by mammalian astrocytes in vivo and is the first direct comparison between the astrocyte, neuron, and oligodendrocyte transcriptomes. We demonstrate that Aldh1L1, a highly expressed astrocyte gene, is a highly specific antigenic marker for astrocytes with a substantially broader, and therefore potentially more useful, pattern of astrocyte expression than the traditional astrocyte marker GFAP. This transcriptome database of acutely isolated and highly pure populations of astrocytes, neurons and oligodendrocytes provides a resource to the neuroscience community by providing improved cell type specific markers and for better understanding of neural development, function, and disease. We acutely purified mouse astrocytes from early postnatal ages (P1) to later postnatal ages (P30), when astrocyte differentiation is morphologically complete (Bushong et al., 2004), and acutely purified mouse OL-lineage cells from stages ranging from OPCs to newly differentiated OLs to myelinating OLs. We extracted RNA from each of these highly purified, acutely isolated cell types and used GeneChip Arrays to determine the expression levels of over 20,000 genes and construct a comprehensive database of cell type specific gene expression in the mouse forebrain. Analysis of this database confirms cell type specific expression of many well characterized and functionally important genes. In addition, we have identified thousands of new cell type enriched genes, thereby providing important new information about astrocyte, OL, and neuron interactions, metabolism, development, and function. This database provides a comparison of the genome-wide transcriptional profiles of the main CNS cell types and is a resource to the neuroscience community for better understanding the development, physiology, and pathology of the CNS. Keywords: Developmental CNS Cell type comparision FACS purification of astrocytes: Dissociated forebrains from S100β-EGFP mice were resuspended in panning buffer (DBPS containing 0.02% BSA and 12.5 U/ml DNase) and sequentially incubated on the following panning plates: secondary antibody only plate to deplete microglia, O4 plate to deplete OLs, PDGFRα plate to deplete OPCs, and a second O4 plate to deplete any remaining OLs. This procedure was sufficient to deplete all OL-lineage cells from animals P8 and younger, however, in older animals that had begun to myelinate, additional depletion of OLs and myelin debris was accomplished as follows. The nonadherent cells from the last O4 dish were harvested by centrifugation, and the cells were resuspended in panning buffer containing GalC, MOG, and O1 supernatant and incubated for 15 minutes at room temperature. The cell suspension was washed and then resuspended in panning buffer containing 20 μg donkey anti-mouse APC for 15 minutes. The cells were washed and resuspended in panning buffer containing propidium iodide (PI). EGFP+ astrocytes were then purified by fluorescence activated cell sorting (FACS). Dead cells were gated out using high PI staining and forward light scatter. Astrocytes were identified based on high EGFP fluorescence and negative APC fluorescence from indirect immunostaining for OL markers GalC, MOG, and O1. Cells were sorted twice and routinely yielded >99.5% purity based on reanalysis of double sorted cells.; FACS purification of neurons: EGFP- cells were the remaining forebrain cells after microglia, OLs, and astrocytes had been removed, and were primarily composed of neurons, and to a lesser extent, endothelial cells (we estimate < 4% endothelial cells at P7 and < 20% endothelial cells at P17). EGFP- cells from S100β-EGFP dissociated forebrain were FACS purified in parallel with astrocyte purification and were sorted based on their negative EGFP fluorescence immunofluorescence. Cells were sorted twice and routinely yielded >99.9% purity. In independent preparations, the EGFP- cell population was additionally depleted of endothelial cells and pericytes by sequentially labeling with biotin-BSL1 lectin and streptavidin-APC while also labeling for OL markers as described above. Cells were sorted twice and routinely yielded >99.9% purity.; Panning purification of oligodendrocyte lineage cells: Dissociated mouse forebrains were resuspended in panning buffer. In order to deplete microglia, the single-cell suspension was sequentially panned on four BSL1 panning plates. The cell suspension was then sequentially incubated on two PDGFRα plates (to purify and deplete OPCs), one A2B5 plate (to deplete any remaining OPCs), two MOG plates (to purify and deplete myelinating OLs), and one GalC plate (to purify the remaining PDGFRα-, MOG-, OLs). The adherent cells on the first PDGFRα, MOG, and GalC plates were washed to remove all antigen-negative nonadherent cells. The cells were then lysed while still attached to the panning plate with Qiagen RLT lysis buffer, and total RNA was purified. Purified OPCs were >95% NG2 positive and 0% MOG positive. Purified Myelin OLs were 100% MOG positive, >95% MBP positive, and 0% NG2 positive. Purified GalC OLs depleted of OPCs and Myelin OLs were <10% MOG positive and ~50% weakly NG2 positive, a reflection of their recent development as early OLs.; Data normalization and analysis: Raw image files were processed using Affymetrix GCOS and the MAS 5.0 algorithm. Intensity data was normalized per chip to a target intensity TGT value of 500, and expression data and absent/present calls for individual probe sets were determined. Gene expression values were normalized and modeled across arrays using the dChip software package with invariant-set normalization and a PM model. (www.dchip.org, Li and Wong, 2001). The 29 samples were grouped into 9 sample types: Astros P7-P8, Astros P17, Astros P17-gray matter (P17g), Neurons P7, Neurons P17, Neurons-endothelial cell depleted (P7n, P17n), OPCs, GalC-OLs, and MOG-OLs. Gene filtering was performed to select probe sets that were consistently expressed in at least one cell type, where consistently expressed was defined as being called present and having a MAS 5.0 intensity level greater than 200 in at least two-thirds of the samples in the cell type. We identified 20,932 of the 45,037 probe sets that were consistently expressed in at least one of the nine cell types. The Significance Analysis of Microarrays (SAM) method (Tusher et al., 2001) was used to determine genes that were significantly differentially expressed between different cell types (see Supplemental Table S2 for SAM cell type groupings). Clustering was performed using the hclust method with complete linkage in R. Expression values were transformed for clustering by computing a mean expression value for the gene using those samples in the corresponding SAM statistical analysis, and then subtracting the mean from expression intensities. In order to preserve the log2 scale of the data, unless otherwise indicated, no normalization by variance was performed. Plots were created using the gplots package in R. The Bioconductor software package (Gentleman et al., 2004) was used throughout the expression analyses. Functional analyses were performed through the use of Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com).
2007-12-30 | E-GEOD-9566 | biostudies-arrayexpress
Project description:Characterizing the developmental trajectory of oligodendrocyte progenitor cells (OPC) is of great interest given the importance of these cells in the remyelination process. However, studiescof human OPC development remain limited by the availability of whole cell samples and material that encompasses a wide age range, including time of peak myelination. In this study, we apply single cell RNA sequencing to viable whole cells across the age span and link transcriptomic signatures of oligodendrocyte-lineage cells with stage-specific functional properties. Cells were isolated from surgical tissue samples of second-trimester fetal, 2 year old pediatric, 13 year old adolescent, and adult donors by mechanical and enzymatic digestion, followed by percoll gradient centrifugation. Gene expression was analyzed using droplet-based RNA sequencing (10X Chromium). Louvain clustering analysis identified 3 distinct cellular subpopulations based on 5613 genes, comprised of an early OL precursor cell (e-OPC) group, a late OPC group (l-OPC), and a mature OL (MOL) group. Gene ontology terms enriched for e-OPCs were cell cycle and development, for l-OPCs were extracellular matrix and cell adhesion, and for MOLs were myelination and cytoskeleton. The e-OPCs were mostly confined to the premyelinating fetal group, and the l-OPCs were most highly represented in the pediatric age group, corresponding to the peak age of myelination. Cells expressing a signature characteristic of l-OPCs were identified in the adult brain in situ using RNAScope. These findings highlight the transcriptomic variability in OL-lineage cells before, during, and after peak myelination and contribute to identifying novel pathways required to achieve remyelination.
2021-01-12 | GSE155488 | GEO
Project description:This study addresses the mechanisms regulating the identity of oligodendrocyte progenitor cells (OPC) in physiological conditions and the pathological mechanisms leading to autosomal adrenoleukodystrophy (ADLD), a disease caused by persistence of lamin B1 (LMNB1) expression in oligodendrocytes (OL). Using mouse primary OPCs and a novel technique for the study of DNA-protein interaction, we identify the genomic regions showing dynamic interactions with LMNB1. The transition from ESC to OPC revealed increased association of gene involved in pluripotency and neuronal function and decreased association of gene categories associated with the differentiation of OPC into OL (including genes regulating epigenetic modifiers, RNA processing and protein phosphorylation). Since LMNB1 declines during the transition from OPC to OL, and its persistent expression has been linked to pathological conditions, such as ADLD, we further identified the molecular maps of genome-lamina reorganization in OPC differentiating into OL which continue to express LMNB1 (mOLLMNB1). The transition from OPC to mOLLMNB1 revealed increased association to LMNB1 of genomic regions encoding for lipid metabolism and protein phosphorylation, and decreased association of genes negatively regulating differentiation. These data highlight the importance of LMNB1 in physiological conditions and identify mechanisms responsible for late onset ADLD.
2020-03-16 | GSE135834 | GEO
Project description:In the central nervous system (CNS), myelin formation by oligodendrocytes (OLs) relies on actin dynamics. Actin polymerization supports the ensheathment step, when the OL process contacts and surrounds a selected portion of axon. While a drastic shift to actin depolymerization is then required to enable the wrapping process and the formation of a multilayered myelin sheath. Molecular mechanisms regulating this switch to actin depolymerization are not fully elucidated. P21-activated kinase 1 (PAK1), a downstream effector of Rho GTPases Rac1 and Cdc42, highly expressed in OLs, can regulate actin dynamics through its kinase activity. We found that PAK1 loss-of-function in OLs, in vivo, stimulates the wrapping and the thickening of myelin sheaths. We showed that PAK1 is highly expressed in myelinating OLs, yet in its inactive form, the one with the ability to trigger actin disassembly. Interestingly, we found that modulations of PAK1 kinase activity control actin dynamics in OLs, thereby myelin formation. Our data demonstrate that PAK1 inactivation in OLs is required for actin depolymerization and proper myelin formation, suggesting the presence of an endogenous PAK1 inhibitor in myelinating OLs. Proteomic analysis of PAK1 binding partners enabled us to identify several proteins likely to regulate its activity.
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