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Single Cell RNA-sequencing of murine tail interfollicular epidermis basal cells at postnatal days 7, 30 and in adult

ABSTRACT: The purpose of this study was to define, at the single cell level, the transcriptionnal profile of murine tail epidermal basal cells during potsnatal growth and to compare with adult homeostatic basal cells. The analysis was performed on Lgr5DTR-EGFP mice (Tian et al., 2011)(knockin mice containing an Enhanced Green Fluorescent Protein (EGFP) under the control of the Lgr5 regulatory region), allowing to identify and exclude Lgr5-expressing cells of the bulge and basal cells of the interfollicular epidermis were enriched using EGFP negative, CD34 negative, alpha6 integrin positive gating. Our single-cell RNA sequencing revealed that epidermal basal cells at P7 form a more uniform population compared to adult basal cells and that heterogeneity starts to appear around P30.

ORGANISM(S): Mus musculus

PROVIDER: GSE146122 | GEO | 2020/04/11

REPOSITORIES: GEO

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Project description:The skin interfollicular epidermis (IFE) is the first barrier against the external environment and its maintenance is critical for survival. Two seemingly opposite theories have been proposed to explain IFE homeostasis. One posits that IFE is maintained by a long-lived slow-cycling stem cell (SC) population that give rise to short-lived transit-amplifying (TA) cell progeny, while the other suggests that homeostasis is achieved by a single committed progenitor (CP) that balances stochastic fate. Here, we probed the cellular heterogeneity within the IFE using two different inducible CREER targeting IFE progenitors. Quantitative analysis of clonal fate data and proliferation dynamics demonstrate the existence of two distinct proliferative cell compartments composed of slow-cycling SC and CP, both of which undergo population asymmetric self-renewal. However, following wounding, only SCs contribute substantially to the repair and long-term regeneration of the tissue, while CP cells make a minimal and transient contribution. Transcriptional profile of InvCREER/RosaYFP and K14CREER/RosaYFP targeted FACS-isolated alpha6 integrin-high CD34-neg basal cells from mouse tail epidermis. Skin epidermis was removed from tail bone and incubated overnight in HBSS (Gibco) 0.25% trypsin (Gibco) at 4M-BM-0C. Epidermis was separated from the dermis and incubated on a rocking plate (100 rpm) at room temperature for 5 min. Basal cells were mechanically separated from the epidermis by flushing 10 times under the epidermis. Tissues were then cut in pieces of 1 mm2 with scalpel, and trypsin was neutralized by adding DMEM medium (Gibco) supplemented with 2% Chelex Fetal Calf Serum (FCS). Samples were filtrated on 70 and 40M-BM-5m filter (Falcon). Immunostaining was performed using biotin-conjugated anti-CD34 (clone RAM34; BD Biosciences) PE-conjugated anti-M-NM-16-integrin (clone GoH3; BD biosciences). Primary antibodies were washed with 2% FCS/PBS and cells were incubated for 30 min in APC conjugated streptavidin (BD Biosciences) secondary antibodies, on ice, with shaking every 10 min. Living K14- and involucrin-expressing epidermal cells were gated by forward scatter, side scatter, negative staining for Hoechst dye and by following the YFP signal. Basal cells from the interfollicular epidermis were targeted using CD34 negative alpha 6 high gating. Fluorescence-activated cell sorting analysis was performed using FACSAria I at high pressure (70 psi) and FACSDiva software (BD Biosciences). Sorted cells were harvested directly in the lysis buffer provided by the RNeasy microkit (QIAGEN) supplemented with 1M-BM-5l of beta-mercaptoethanol for every 100M-BM-5l of lysis buffer. RNA extraction was performed on freshly sorted cells according to the manufacturerM-bM-^@M-^Ys protocol.

Project description:Epidermal stem cells ensure that skin homeostasis is maintained. In murine skin, epidermal stem cells cluster at specific niches where, under steady-state conditions, they undergo cycles of dormancy and activation1. When cellular replenishment is required, epidermal stem cells egress from the niche and proliferate for a limited number of times to subsequently feed into the differentiated compartment1-3. However, only a subset of stem cells becomes active during each round of morphogenesis, suggesting that stem cells coexist in heterogeneous responsive states within the same niche. Using a circadian clock fluorescent reporter mouse model, we show that the dormant epidermal stem cell niche contains two coexisting populations of stem cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. In dormant niches, the core molecular clock protein Bmal1 transcriptionally modulates the expression of stem cell regulatory genes, including modulators of Wnt and TGFb, to create two coexisting stem cell populations, one predisposed, and the other less prone, to activation. Unbalancing this equilibrium of epidermal stem cells, through conditional epidermal deletion of Bmal1, resulted in a long-term progressive accumulation of non-responsive stem cells, premature impairment of tissue self-renewal, and a significant reduction in the development of squamous cell carcinomas. Our results indicate that the molecular clock machinery fine-tunes the spatiotemporal behavior of epidermal stem cells within their niche, and that perturbation of this mechanism affects tissue homeostasis and the predisposition to neoplastic transformation. The goals of this study was to compare the transcriptome of epidermal stem cells according to their circadian rhythm phase. We isolated epidermal stem cells (bulge cells; alpha6bright/CD34+ population) from 19 days old Per1-Venus mice and separated them according to Venusbright (clock positive) and Venus dim (clock negative). The goals of this study was to compare the transcriptome of epidermal stem cells in which their circadian rhythm machinery has been perturbed by deleting the gene that encodes for Bmal1. We compared the transcriptomes of basal interfollicular epidermis cells (alpha6 integrin bright/CD34- cells) from the dorsal skin of 1 year old BmalKO mice and their respective control littermates. Each array corresponds to purified cells from approximately 5 mice. We profiled three samples of Venus bright and three of Venus dim epidermal stem cells. Each sample consisted of epidermal stem cells isolated from aproximately 20 mice (in order to obtain enough number of cells to perform high quality arrays). We profiled three WT samples and 3 KO samples. Each sample corresponds to basal interfollicular epidermis cells purified from 5 mice.

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Single Cell RNA-sequencing of murine tail interfollicular epidermis basal cells at postnatal days 7, 30 and in adult

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