Understanding the role of microRNAs in astrocyte development
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Shen-Yi Howng
PROVIDER: E-MTAB-2995 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Transcription profiling by array of wild-type or homozygous dicer1 conditional knockout forebrain P7 Aldh1l1-EGFP cells and Ald1l1-EGFP positive primary astrocytes
2015-02-13 | E-MTAB-2995 | ExpressionAtlas
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:BAC array purification of hippocampus astroglial ribosome-bound transcriptome in astrocytes from Aldh1l1:Rpl10a eGFP/ Cx43 KO and Aldh1l1:Rpl10a eGFP/ Cx43FL mice
2019-05-22 | GSE110721 | GEO
Project description:The transcription factor Pax6 is comprised of the paired domain (PD) and homeodomain (HD). In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific subpopulations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification. Pax6 also regulates the entire lens developmental program. To reconstruct Pax6-dependent gene regulatory networks (GRNs), ChIP-seq studies were performed using lens and forebrain chromatin from mice. A total of 3,723 (forebrain) and 3,514 (lens) Pax6-containing peaks were identified, with ~70% of them found in both tissues and thereafter called “common” peaks. Analysis of Pax6-bound peaks identified motifs that closely resemble Pax6-PD, -PD/HD and -HD established binding sequences. Mapping of H3K4me1, H3K4me3, H3K27ac, H3K27me3 and RNA polymerase II revealed distinct types of tissue-specific enhancers bound by Pax6. Pax6 directly regulates cortical neurogenesis through activation (e.g. Dmrta1 and Ngn2) and repression (e.g. Ascl1, Fezf2, and Gsx2) of transcription factors. In lens, Pax6 directly regulates cell cycle exit control via components of FGF (Fgfr2, Ccnd1, and Prox1) and Wnt (Dkk3, Wnt7a, Lrp6, Bcl9l, and Ccnd1) signaling pathways. Collectively, these studies provide genome-wide analysis of Pax6-dependent GRNs in lens and forebrain and establish novel roles of Pax6 in organogenesis. Examination of Pax6 in mouse embryonic forebrain and newborn lens. We performed ChIP-seq on mouse E12.5 embryonic forebrain and newborn lens. Genome-wide binding sites of Pax6, H3K4me1, H3K4me3, H3K27ac, H3K27me3, and Pol2 were generated. We also performed RNA-seq on mouse E12.5 embryonic forebrain and newborn lens epithelial cells and fibers.
2015-07-09 | E-GEOD-66961 | biostudies-arrayexpress
Project description:Aldh1l1-EGFP cells were FACS and isolated from postnatal day 7 mouse forebrains. Total RNAs were then isolated and analyzed for microRNA expression.
2015-02-24 | E-MTAB-3336 | biostudies-arrayexpress
Project description:Study of Sox18 regulated genes: Human umbilical vein endothelial cells (HUVEC) were either transduced with adenoviral vectors expressing SOX18 from an IRES-EGFP casette, or IRES-EGFP alone, or left untreated. After 16 hours, mRNA was isolated and analyzed by hybridization to Affymetrix HG-U133A arrays. Human umbilical vein endothelial cells (HUVEC) were either transduced with adenoviral vectors expressing SOX18 from an IRES-EGFP casette, or IRES-EGFP alone, or left untreated. After 16 hours, mRNA was isolated and analyzed by hybridization to Affymetrix HG-U133A arrays.
2012-01-23 | E-GEOD-8952 | biostudies-arrayexpress
Project description:Analysis of human liver cell line (Huh7) infected with HEV ORF3 expressing (Ad-orf3-egfp) and control recombinant adenovirus (Ad-egfp). Hepatitis E virus ORF3 protein (pORF3) is known to modulate the host cell. Results provide insight into the role of this protein for HEV infection and pathogenesis. The gene expression experiment of Huh7 hepatoma cell line infected with ORF3 expressing recombinant adenoviruses (Ad-orf3-egfp) and with control recombinant adenoviruses (Ad-egfp) was performed in replicates of three.
2010-12-01 | E-GEOD-23412 | biostudies-arrayexpress
Project description:To identify factors responsible for the differential localization and activity of PIK3C2Bin the presence or absence of serum mitogens we took a quantitative functional proteomics approach based on stable isotope labeling of amino acids in cell culture (SILAC). Genome-engineered HEK293T cells endogenously expressing eGFP-PI3KC15were cultured in serum-containing or serum-deprived media containing heavy or light amino acids, and subjected to affinity capture using a GFP trap matrix and LC-MS/MS
2019-10-02 | PXD014533 | Pride
Project description:We provide an annotated cDNA clone collection which is particularly suitable for transcriptomic analysis in the mouse brain. Using it on microarrays, we compared the transcriptome of EGFP positive and negative cells in a parvalbumin-egfp transgenic background and showed that more than 30 % of clones are differentially expressed. Our clone collection will be a useful resource for the study of the transcriptome of single cell types. Keywords: Cell type comparison Comparison between fluorescent and non-fluorescent cells isolated from the visual cortex of parvalbumin-egfp transgenic mice. Using five biological replicates with a dye-swap strategy (10 hybridisations).
2008-08-22 | E-GEOD-8968 | biostudies-arrayexpress
Project description:We have generated transgenic mice with tetracycline-regulated conditional expression of a constitutively active allele of FoxO3 under the control of the forebrain-specific CaMKIIa promoter. In adult animals, there was a reduction of brain weight by 30% and an almost complete loss of the dorsal dentate gyrus with normal cortical layering. Interestingly, the adult mice showed motor hyperactivity and a selective loss of long-term memory with normal spatial learning. We observed enhanced apoptosis starting from day E10.5. Performing microarray expression analyses and Q-PCR validation with E12.5 forebrain RNA, we observed an over-representation of thalamic markers and an under-representation of cortical markers in transgenic as compared to control animals. Immunohistochemical data show a loss of progenitors in the lateral ventricles. Up-regulation of Pik3ip1 as a target gene of FoxO3 could be responsible for the observed increase in apoptosis. The obtained forebrain expression signature is reminiscent of a Pax6 knockdown phenotype showing that expression of this FoxO3 allele during development affected neural progenitor survival and overall brain development. Conclusion: Neural progenitors are vulnerable to constitutively active FoxO3-induced apoptosis. We sought to determine the transcriptional differences in forebrains from E12.5 mice expressing a constitutively active alleleof FoxO3 under the control of the forebrain-specific CaMKIIa promoter. To this end two time-pregnant dams were sacrificed 12 days after the vaginal plug was detected, and the embryos were prepared. Visual staging of the embryos confirmed their age. Genotyping and luciferase measurements were performed in order to assess presence and acitivity of the transgenes.
2012-09-13 | E-GEOD-21089 | biostudies-arrayexpress