Gene expression profiling of infrequently and frequently dividing cells in the mouse epidermis
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Aiko Sada
PROVIDER: E-GEOD-70523 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Quiescent hair follicle (HF) bulge stem cells (SCs) differentiate to early progenitor (EP) hair germ (HG) cells, which divide to produce transit-amplifying (TA) matrix cells. EPs can revert to SCs upon injury, but whether this de-differentiation occurs in normal HF homeostasis (hair cycle), and the mechanisms regulating both differentiation and de-differentiation are unclear. Here we use lineage tracing, gain of function, transcriptional profiling, and functional assays to examine the role of observed endogenous Runx1 level changes in the hair cycle. We find that forced Runx1 expression implements hair degeneration (catagen) and simultaneously promotes changes in the quiescent bulge SC transcriptome towards a cell-state resembling the EP HG fate. This cell-state transition is functionally reversible. We propose that SC differentiation and de-differentiation are likely to occur during normal HF degeneration and niche restructuring in response to changes in endogenous Runx1 levels associated with SC location with respect to the niche. Freshly isolated skin cells were FACS sorted based on K15-GFP+/a6-integrin+/CD34- as hair germ and K15-GFP+/a6-integrin+/CD34+ bulge cells. Duplicate samlpes from mice at PD20 that showed telogen morphology throughout skin were used for RNA prepartion and Affymetrix analysis. Wild and transgenic samples after 1-day of doxycycline treatement were compared.
2014-01-15 | E-GEOD-53077 | biostudies-arrayexpress
Project description:In homeostasis of adult vertebrate tissues, stem cells are thought to self-renew by infrequent and asymmetric divisions that generate another stem cell daughter and a progenitor daughter cell committed to differentiate. This model is based largely on in vivo invertebrate or in vitro mammal studies. Here we examine the dynamic behaviour of adult hair follicle stem cells in their normal setting by employing mice with repressible H2B-GFP expression to track cell divisions and Cre inducible mice to perform long-term single cell lineage tracing. We provide direct evidence for the infrequent stem cell division model in intact tissue. Moreover, we find that differentiation of progenitor cells occurs at different times and tissue locations than self-renewal of stem cells. Distinct fates of differentiation or self-renewal are assigned to individual cells in a temporal-spatial manner. We propose that large clusters of tissue stem cells behave as populations, whose maintenance involves unidirectional daughter-cell fate decisions. We used microarrays to expression profile the stem cell progeny generated at distinct differentiation and self-renewal stages. Experiment Overall Design: We employed double transgenic mice, K5tTA x pTRE-H2B-GFP in which an epithelial Keratin 5 (K5) promoter drove repressible histone H2B-GFP expression. Repression is achieved by feeding the mice doxycycline for a period of time (chase), when the H2B-GFP dilutes in cells by 2-fold at division. This allowed us to quantify precise proliferation history in vivo, from the amount of H2B-GFP fluorescence retained in cells after chase. After various chase periods we sacrificed mice at different ages corresponding to distinct phases of hair cycle. We isolated skin cell suspensions from these mice and stained them for surface expression of stem cell niche bulge(Bu) markers CD34 and alpha 6-integrin. Fluorescence-activated cell sorting (FACS) revealed histograms with distinct peaks of 2-fold median H2B-GFP intensity corresponding to distinct divisions. For microarray, we FACS sorted bulge(Bu) and non-bulge(NonBu) cells based cell surface marker staining and their proliferation history with H2B-GFP intensity, after two doxycycline chase schemes (Postnatal Day 18-PD21 or PD22-PD25), then extract total RNA from each cell fractions. For microarrays 5 ng of high quality RNA (Bioanalyzer, Agilent) were amplified (Ovation Amplification; Nugene) in the Cornell Microarray Core Facility. GeneChip IVT labelling was followed by Gene Chip MOE 430 2.0 hybridization, GeneArray 3000 scanning, GCOS generation of present calls and signal values (Affymetrix).
2009-09-07 | E-GEOD-16516 | biostudies-arrayexpress
Project description:Infrequently (LRCs) and frequently (non-LRCs) dividing cells in the mouse epidermis are moleculary distinct.
2016-09-01 | GSE70523 | GEO
Project description:Runt domain (Runx) transcription factors constitute a metazoan family of sequence specific DNA binding proteins that are essential for animal development. The sea urchin Runx gene SpRunt-1, which is expressed globally during early embryogenesis, is required for blastula stage cell proliferation and subsequently for cell survival. To obtain a comprehensive list of SpRunt-1 regulatory targets we screened a custom microarray representing the blastula stage transcriptome of Strongylocentrotus purpuratus, comparing relative hybridization signals generated by labeled RNA from 18 hr control versus SpRunt-1 morphant embryos. For an 8x15k chip the 8 arrays were 4 biological replicates and a dye swap of the same 4. Biological Replicates - Four separate embryo cultures, each from a different mating pair were initiated and divided into a control group and a treatment group. All of the embryos in the treatment group were micoinjected with a splice-blocking morpholino antisense oligonucleotide (MASO) targeted to SpRunt-1. All control and sample embryos were developed to hatched blastula stage (18 hours post-fertilization) in filtered seawater at 15°C. Total RNA was extracted from each of the four separate samples (3,600 embryos in each sample) with an RNA yield near 5 micro grams per sample. Total RNA was also extracted from the untreated embryos of each separate culture pooled together (about 14,400) with an RNA yield near 20 micro grams. Technical (dye swap) Replicates - Control derived Cy3 (green) labeled aRNA was mixed with sample derived Cy5 (red) labeled aRNA in one biological replicate set of 4 samples and using half of the extracted RNA amount. The other half of the RNA was reciprocally labeled with control derived aRNA Cy5 labeled and sample derived aRNA Cy3 labeled.
2010-05-16 | E-GEOD-19751 | biostudies-arrayexpress
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare palatal epithelial basal cell transcriptomes between infrequently and frequently dividing cells (IDC/FDCs). Methods: RNA was isolated from 200-1000 palatal basal epithelial cells and libraries prepared using the SoLo RNA-Seq library preparation kit (NuGEN). mRNA profiles of 14-day doxycycline chased K5-tTA;tetO-H2B-GFP (containing populations of slow-cycling GFP-HI and fast-cycling GFP-LO) mice were generated with 6 matched samples using an Illumina HiSeq4000. RNA Sequencing Allows for Differential Gene Expression Analysis of infrequently dividing and frequently dividing cells (IDC/FDC). RNA-seq fastq files were trimmed and aligned to the latest mouse genome, mm10 (GRCm38/GCA_000001635.2) using BBMap. A matrix of the data was generated using featureCounts. RNAseq data was analyzed with DESeq2. All statistical analyses were performed using R version 3.4.1 and RStudio version 1.0.143. Validation was performed using lineage tracing and immunofluorescence immunohistochemistry. Results: Using an optimized data analysis workflow, approximately 2% of the transcripts showed differential expression between the GFP-HI and GFO-LO palatal basal epithelial cell with a p value <0.05. Analysis of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to oral epithelial stem cell, such as Lrig1 and Igfbp5 as followed up on in the associated manuscript. Conclusions: Our study represents the first detailed analysis of palatal basal epithelial stem cell transcriptomes, with biologic replicates, generated by RNA-seq technology. Our results show GFP-LO infrequently dividing cells are express a distinct transcriptional profile compared to frequently dividing neighbor cells such as Lrig1 and Igfbp5.
2019-10-22 | GSE139146 | GEO
Project description:Woodchucks that were infected with WHC virus developed HCCs. HCCs and surrounding tissues were removed and RNA was extracted from microarray study. Human, rat and mouse arrays were used due to the lack of a woodchuck array. Same procedures and parameters were used to conduct this experiment. Experiment Overall Design: 3 woodchucks with HCCs were used. HCCs and their surrouding normal tissues were harvested. These 3 pairs of tissues were cross-species hybridized into a human, rat and mouse chip under same conditions. Data analysis were performed to compare the results.
2008-06-11 | E-GEOD-11050 | biostudies-arrayexpress
Project description:We have used the slow cycling property, found in hair follicle stem cells, to look for LRCs in sweat glands as putative stem cells. Gene expression profiling allowed us to determine the common and unique genes expressed in SG LRCs and non-LRCs. This allowed us to probe for the signaling pathways active in this skin appendage as well as determine potential molecular markers of SG LRCs.
2013-12-24 | GSE49011 | GEO
Project description:We have used the slow cycling property, found in hair follicle stem cells, to look for LRCs in sweat glands as putative stem cells. Gene expression profiling allowed us to determine the common and unique genes expressed in SG LRCs and non-LRCs. This allowed us to probe for the signaling pathways active in this skin appendage as well as determine potential molecular markers of SG LRCs. Sweat gland label retaining cells (LRCs) and surrounding basal layer cells were isolated from K5TetOffTreH2BGFP mice after a 4 week chase with doxycycline and compared to the basal layer of the sole's epidermis in 2 independent experiments.
2013-12-24 | E-GEOD-49011 | biostudies-arrayexpress
Project description:Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells diminished tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of BCSCs (ESA+-CD44+-CD24-) and the tumor-differentiated cells (ESA+-CD44+-CD24+) from MCF7-Tet-On-SrcDN. ESA+-CD44+-CD24- grew in suspension forming mammospheres, and producing tumors in nude mice, while ESA+-CD44+-CD24+ were poorly/non-tumorigenic. Doxycycline-induction of SrcDN inhibited BCSC tumorigenesis, selfrenewal, and stem-cell markers expression. SrcDN significantly inhibited SFE, and stem-cell markers expression in triple-negative breast cancer (TNBC) MDA-MB-231 and SUM159PT cells. Inducible depletion of c-Src caused similar effects in MDA-MB-231 cells. In MCF7-Tet-On-SrcDN derived mammospheres SrcDN-induction inhibited expression, and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, tumorigenicity, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.
2020-07-23 | PXD017789 | Pride
Project description:Transcriptional coactivator Mediator complex facilitates transcription of various transcription factors. Previously, we have generated Med1 conditional null mice, where a critical subunit of Mediator, Med1, is removed from keratinocytes. Here we present evidence that ablation of Med1 accelerated epidermal regeneration after injury. As bulge keratinocyte stem cells are important contributors to regenerate epidermis, we first analyzed properties of stem cells in Med1 null mice. BrdU long retaining analysis revealed that deletion of Med1 still maintained quiescence of bulge keratinocyte stem cells, despite of general hyperplasia observed in Med1 deficient keratinocytes. Gene expression analysis demonstrated that a series of niche matrix proteins decreased in Med1 deficient keratinocytes. In contrast, the expression of stem cell marker Sox9 was not altered, suggesting stem cells are present but activated because of abnormal niche surrounding stem cells. In addition, Med1 deletion suppressed injury induced inflammatory reaction, which indirectly regulates epidermal regeneration. We also indicated that TGFβ1 significantly decreased in both bulge and epidermal keratinocytes upon Med1 deletion. Our study demonstrates that coactivator Med1 has a critical role to maintain bulge stem cells and epidermal regeneration presumably through regulation in TGFβ signaling. n=3 WT and KO (each sample contain RNA isolated from wounded or nonwounded skins excised from 3 mice)
2014-01-01 | E-GEOD-50670 | biostudies-arrayexpress