Determination of mRNA half-lives in embryonic mouse skin
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2500
ORGANISM(S): Mus musculus
SUBMITTER: Denis Headon
PROVIDER: E-MTAB-5841 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined due to a lack of functional subclasses. Here we reveal the presence of multiple lineages of dermal fibroblasts within the dorsal back. Genetic lineage tracing and transplantation assays demonstrate that the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation is carried out by a single, somitic-derived fibroblast lineage. Reciprocal transplantation of distinct fibroblast lineages between the dorsal back and oral cavity induced ectopic dermal architectures that mimic their place–of-origin. These studies demonstrate that intra and inter-site diversity of dermal architectures are set embryonically and maintained postnatally by distinct lineages of fibroblasts. Lineage-specific cell ablation using transgenic-mediated expression of the simian diphtheria toxin receptor in conjunction with localized administration of diphtheria toxin led to diminished connective tissue deposition in wounds and significantly reduced melanoma growth in the dorsal skin of mice. Using flow cytometry and in silico approaches, we identify CD26/DPP4 as a surface marker that allows for the isolation of this fibrogenic, scar-forming lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. The identification and prospective isolation of these lineages holds promise for translational medicine aimed at in vivo modulation of their fibrogenic behavior. Dermal fibroblasts were harvested for FACS from the oral dermis and cranial dermis of Wnt1Cre; R26mTmG mice, and from ventral and dorsal dermis of En1Cre; R26mTmG mice as previously described. For all microarray analysis, EPFs and ENFs where harvested from backskin of mice at 30 days of age. Positivity for GFP or RFP allowed for the separation of EPFs from ENFs and WPFs from WNFs. RNA was precipitated via chloroform-phenol extraction. Samples were processed for cleanup and concentration using Rneasy MinElute cleanup kit (cat. 74204, QIAGEN). RNA yield was typically 0.5-1 µg RNA/sorted subpopulations. RNA samples from all sorted populations were converted to cDNA using SuperScript III first strand synthesis system for RT-PCR (cat. 18080-051, Invitrogen), and hybridized to Affymetrix Mouse Genome 430 2.0 arrays. Microarrays were normalized by robust multichip average (RMA) and quantile normalization in R. Cluster analysis was performed with AutoSOME (33), using the following settings for gene expression clustering: p-value threshold of 0.05, 100 ensemble iterations, unit variance normalization of arrays, median centering of genes, and sum of squares=1 normalization for both genes and arrays.
2015-05-15 | E-GEOD-65402 | biostudies-arrayexpress
Project description:Increasing evidence suggests that microRNAs may play important roles in regulating self-renewal and differentiation in mammalian stem cells (SCs). Here, we explore this issue in skin. We first characterize microRNA expression profiles of skin SCs versus their committed proliferative progenies and identify a microRNA subset associating with “stemness”. Of these, miR-125b is dramatically downregulated in early SC-progeny. We engineer an inducible mice system and show that when miR-125b is sustained in SC-progenies, tissue balance is reversibly skewed towards stemness at the expense of epidermal, oil-gland and HF differentiation. Using gain-and-loss of function in vitro, we further implicate miR-125b as a repressor of SC differentiation. In vivo, transcripts repressed upon miR-125b induction are enriched >700% for predicted miR-125b targets normally downregulated upon SC-lineage commitment. We verify some of these miR-125b targets, and show that Blimp1 and VDR in particular can account for many tissue imbalances we see when miR-125b is deregulated. We used microarrays to compare the global gene expression profile of early bulge stem cells and non bulge ORS cells. Hair follicle cells were isolated from P4 Backskin of K14-RFP/Sox9-EGFP double transgenic mice as following: interfollicular epidermis sheet was pealed from hair follicle & dermis after dispase treatment. The hair follicle & dermis were first digested by collagenase (Sigma). Intact hair follicles were separated from dermal cells by low speed spinning (20g). The hair follicles were then digested by Trypsin and filtered by 40 µm cell strainers. The isolated hair follicle cells were FACS sorted. Dead cells and large differentiated cells were excluded based on DAPI and side scattering. Early bulge cells were gated as GFPHi,RFPHi. Non-bulge ORS cells were gated as GFP-, RFPHi.
2011-01-04 | E-GEOD-26393 | biostudies-arrayexpress
Project description:Human skin undergoes profound structural remodelling following chronic exposure to ultraviolet radiation (UVR) – photoageing - that is normally studied by comparing photoexposed and photoprotected skin sites. Whilst it is well established that the abundance and distribution of major dermal extracellular matrix (ECM) proteins (principally collagen I and elastin) are compromised in skin, the influence of UVR exposure on other skin protein constituents, including epidermal proteins, is poorly understood. We used label-free LC-MS/MS proteomics to characterise the proteomic profiles of intrinsically aged buttock and photoaged forearm epidermis and dermis. Besides traditional shotgun proteomics we have also developed and used novel proteomics analysis approach to reveal locational changes in peptide yields from proteins named Manchester Peptide Locational Fingerprinting (MPLF) which is based on spectral counting. MPLF is a new proteomic analysis tool that can identify structural modification-associated differences within proteins from label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) datasets generated from biological samples. We have identified a total of 174 dermal proteins and 146 epidermal proteins with MPLF while relative quantification revealed a total of 635 dermal proteins and 926 epidermal proteins significantly different between photoaged forearm and intrinsically-aged buttock according to peak area intensity.
2021-04-09 | PXD021194 | Pride
Project description:To obtain a separation of the epidermal and dermal compartments in order to examine compartment specific biological mechanisms in the skin we incubated 4 mm human skin punch biopsies in ammonium thiocyanate (NH4SCN). We wanted to test 1) the histological quality of the dermo-epidermal separation obtained by different incubation times 2) the amount and quality of extractable epidermal RNA, and 3) its impact on sample RNA expression profiles assessed by large-scale gene expression microarray analysis in both normal and inflamed skin. At 30 minutes incubation, the split between dermis and epidermis was not always histologically well-defined (i.e. occurred partly intra-epidermally) but varied between subjects. Consequently, curettage along the dermal surface of the biopsy was added to the procedure. This modified method resulted in an almost perfect separation of the epidermal and dermal compartments and satisfactory amounts of high-quality RNA were obtained. Hybridization to Affymetrix HG_U133A 2.0 GeneChips showed that ammonium thiocyanate incubation had a minute effect on gene expression resulting in only one significantly downregulated gene (cystatin E/M). We conclude that epidermis can be reproducibly and almost completely separated from the dermis of 4 mm skin biopsies by 30 min incubation in 3.8% ammonium thiocyanate combined with curettage of the dermal surface, producing high-quality RNA suitable for transcriptional analysis. Our refined method of dermo-epidermal separation will undoubtedly prove valuable in the many different settings, where the epidermal and dermal compartments need to be evaluated separately. Upper buttock skin in 4 healthy subjects was exposed to sodium lauryl sulphate, or sampled directly. For each subject, 4 biopsies were obtained: Two from inflamed skin, and two from adjacent normal skin. One irritated and one normal skin sample was placed directly in RNAlater. The remaining two samples were incubated in ammonium thiocyanate for 30 minutes at RT and then placed in RNAlater without performing any separation of the dermal and epidermal layers. This was done to investigate the effect of 30 minutes treatment with ammonium thiocyanate on both inflamed and non-inflamed skin. Data was normalized with quantile method (matrix 1) Forearm biopsies from 13 volunteers were separated to epidermis and dermis by use of ammonium thiocyanate. For comparison of full skin and epidermis without irritation, data from identical probe sets from HG_U133A 2.0 and HG_U133 plus 2.0 was extracted and normalised as one data set using quantile method (matrix 2).
2010-03-03 | E-GEOD-15101 | biostudies-arrayexpress
Project description:In this study, we have analyzed DNA methylation changes upon aging of human dermal fibroblasts by using the HumanMethylation27 BeadChip assessing 27,578 unique CpG sites. Cells were isolated from skin samples donated by young (6-23 years) and elderly (60-73 years) patients undergoing surgical interventions. Strikingly, global DNA-methylation profiles of fibroblasts from the same anatomical site clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. Skin samples from younger or elderly donors were treated with dispase (Roche Diagnostics, Mannheim, Germany) for 12 hours to separate the dermis from the epidermis. The dermis was digested with 0,2% collagenase and 1,5% BSA in collagenase buffer (100mM HEPES, 120mM NaCl, 50mM KCl, 1mM CaCl2, 5mM Glucose) for 45 minutes. Dermal remnants were removed by filtering the digest through a 100µm nylon strainer (Falcon, Becton Dickinson [BD], San Jose, USA). The cells were subsequently washed and expanded in standard medium consisting of DMEM (PAA; 1g/L glucose) supplemented with glutamine (PAA), penicillin/sptrepamycin (PAA) and 10% fetal calf serum (Biochrom, Berlin, Germany) in a humidified atmosphere at 5% CO2. Cells were always replated when grown to 80% confluency. For methylation profiles upon aging we have isolated DNA from the samples of passage 3 of younger and elderly donors. For methylation profiles upon long-term culture we have isolated DNA from the samples of early passage (P3) and late passage (P21)
2012-02-14 | E-GEOD-22595 | biostudies-arrayexpress
Project description:The skin plays a crucial role in host defences against microbial attack and the innate cells must provide the immune system with sufficient information to organize these defences. This unique feature makes the skin a promising site for vaccine administration. Although cellular innate immune events during vaccination have been widely studied, initial events remain poorly understood. Our aim is to determine molecular biomarkers of skin innate reaction after intradermal (i.d.) immunization. Using an ex vivo human explant model from healthy donors, we investigated by NanoLC-MS/MS analysis and MALDI-MSI imaging, to detect innate molecular events (lipids, metabolites, proteins) few hours after i.d. administration of seasonal trivalent influenza vaccine (TIV). This multimodel approach allowed to identify early molecules differentially expressed in dermal and epidermal layers at 4 and 18 h after TIV immunization compared with control PBS. In the dermis, the most relevant network of proteins upregulated were related to cell-to-cell signaling and cell trafficking. The molecular signatures detected were associated with chemokines such as CXCL8, a chemoattractant of neutrophils. In the epidermis, the most relevant networks were associated with activation of antigen-presenting cells and related to CXCL10. Our study proposes a novel step-forward approach to identify biomarkers of skin innate reaction.
2020-01-29 | PXD017313 | Pride
Project description:Characterization of swine skin DC subpopulations by means of global expression analysis. Three sets of swine skin DC plus peripheral blood B cells and monocytes (1 color hybridizations)
2014-12-19 | E-GEOD-53500 | biostudies-arrayexpress
Project description:The morphology and the behavior of skin and oral tissue keratinocytes are different. One significant dissimilarity between the two sites is the response to injury. Oral and skin keratinocytes have intrinsic differences in the response to injury and such differences are reflected in gene expression profiles. We used microarrays to investigate differences in global gene expression patterns between baseline skin and oral epithelium sheets without their underlying connective tissue. Paired skin and oral epithelium was separated from the dermis for RNA extraction and hybridization on Affymetrix microarrays. Skin epidermal tissues were obtained from the tail of mice and oral epidermal tissues were obtained from the hard palate. Enzymatically isolated epithelium was used for analysis.
2014-06-15 | E-GEOD-56135 | biostudies-arrayexpress
Project description:Increasing evidence suggests that microRNAs may play important roles in regulating self-renewal and differentiation in mammalian stem cells (SCs). Here, we explore this issue in skin. We first characterize microRNA expression profiles of skin SCs versus their committed proliferative progenies and identify a microRNA subset associating with “stemness”. Of these, miR-125b is dramatically downregulated in early SC-progeny. We engineer an inducible mice system and show that when miR-125b is sustained in SC-progenies, tissue balance is reversibly skewed towards stemness at the expense of epidermal, oil-gland and HF differentiation. Using gain-and-loss of function in vitro, we further implicate miR-125b as a repressor of SC differentiation. In vivo, transcripts repressed upon miR-125b induction are enriched >700% for predicted miR-125b targets normally downregulated upon SC-lineage commitment. We verify some of these miR-125b targets, and show that Blimp1 and VDR in particular can account for many tissue imbalances we see when miR-125b is deregulated. We used microarrays to compare the global miRNA expression profile of P4 stage hair follicle ORS cells from DTG (K14-rtTA,TRE-miR-125b) and control littermates. Hair follicle cells were isolated from P4 Backskin (Dox since P3 for 24hrs) of DTG (K14-rtTA/TRE-miR-125b/K14-H2BGFP), TRE (TRE-miR-125b/K14-H2BGFP), KrtA (K14-rtTA/K14-H2BGFP) as following: interfollicular epidermis sheet was pealed from hair follicle & dermis after dispase treatment.The hair follicle & dermis were first digested by collagenase (Sigma). Intact hair follicles were separated from dermal cells by low speed spinning (20g). The hair follicles were then digested by Trypsin and filtered by 40 µm cell strainers. The isolated hair follicle cells were FACS sorted. During FACS, cells were first gated against CD34 (endothelial cells), CD45 (immune cells), CD114 (melanocytes) and DAPI (dead cells). ORS cells were sorted from the remaining cells as α6HiGFPHi.
2011-01-04 | E-GEOD-26395 | biostudies-arrayexpress
Project description:Cells were generated for a single cell atlas of adult human skin to dissect the cellular and molecular organisation underpinning human skin immune barrier function. Surplus skin from breast reconstruction surgery was collected (n=3), then the top 200 μm layer was taken. Epidermis was separated from the dermis, and both layers were separately digested. Single cells were FACS sorted into eight categories, then loaded onto a 10x Genomics Chromium Controller, and sequenced on an Illumina HiSeq 4000.
2021-02-10 | E-MTAB-8142 | biostudies-arrayexpress