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Single-cell RNA-sequencing of Gli1-positive cells from different compartments of the adult mouse epidermis

ABSTRACT: Single-cell RNA-sequencing of Gli1-positive cells from different compartments of the adult mouse epidermis

PROVIDER: PRJEB30470 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR3013940.fastq.gz	Fastqsanger.gz
	ERR3013950.fastq.gz	Fastqsanger.gz
	ERR3013960.fastq.gz	Fastqsanger.gz
	ERR3013970.fastq.gz	Fastqsanger.gz
	ERR3013980.fastq.gz	Fastqsanger.gz

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Single-cell RNA-sequencing of Gli1-positive cells from different compartments of the adult mouse epidermis

Project description:Tomato-traced single cells from the interfollicular epidermis including the touch dome (Gli1-Tomato+/Sca1+) and the hair follicle (Gli1-Tomato+/Sca1-) were FACS-sorted, randomly sequenced and clustered into epidermal (sub-)populations.

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Expression profile of Gli1-expressing keratinocytes from mouse skin

Project description:To assess if Hedgehog (Hh) responding cells in the skin have a unique expression profile, isolated keratinocytes that express the Hh response gene Gli1 were collected by FACS and their gene expression was compared to sorted CD34-expressing cells from the middle bulge region of the hair follicle and to cells from the interfollicular epidermis (IFE) by hybridization of isolated RNA to gene expression microarrays. Dissociated keratinocytes isolated form back skin of Gli1-eGFP/+ mice (2 mice pooled for each replicate) in adult telogen were sorted based on GFP expression and immunostaining for CD34. Only viable, single cells with immunostaining for α6 Integrin (to mark basal keratinocytes) were collected. GFP(+) cells were collected as the Gli1 cohort. GFP(-) CD34(+) cells were collected as the CD34 cohort, and GFP(-) CD34(-) cells were collected as interfollicular epidermis (IFE) cohort. Total RNA was extracted from each cell population and labeled for hybridization to gene expression microarrays. The experiment was preformed in triplicate, however RNA from the Gli1(+) cells in one replicate was of insufficient quality to analyze. For staining of isolated keratinocytes, cells were incubated on ice for 1 hour in SMEM containing 1%BSA and 15μL of anti-CD49f-PE antibody (BDBioscience) plus 5μL anti-CD34-APC antibody (eBioscience) for each 100μL total volume for each 2 million cells. Immediately prior to sorting, DAPI (Invitrogen) was added to the washed cells at 1μg/mL final concentration.

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Gene expression signature of the three major epidermal differentiation compartments: interfollicular epidermis (IFE), hair follicle (HF) and sebaceous gland (SG).

Project description:There are multiple stem cells in adult mammalian epidermis, but the mechanisms controlling lineage specification are poorly understood. To identify gene expression signatures of the three major epidermal differentiation compartments we micro-dissected individual SG, IFE and HF from adult epidermis. The RNA was isolated from age and sex matched wild-type mice and performed transcriptome analysis with Affymetrix Exon microarrays We micro-dissected individual interfollicular epidermis (IFE), hair follicle (HF) and sebaceous gland (SG) from adult tail epidermis. The RNA was isolated from age and sex matched wild-type mice. Three biological replicates for each epidermal differentiation compartment were analyzed.

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A conditionally immortalized Gli1-positive kidney mesenchymal cell line models myofibroblast transition

Project description:Glioma-associated oncogene homolog-1 (Gli1)-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis, but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KGli1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation, and display robust myofibroblast differentiation upon treatment with transforming growth factor-β (TGF-β). Coculture of KGli1 cells with endothelium stabilizes capillary formation. Single-cell RNA sequencing (scRNA-seq) analysis during differentiation identified autocrine ligand-receptor pair upregulation and a strong focal adhesion pathway signal. This led us to test the serum response factor inhibitor CCG-203971 that potently inhibited TGF-β-induced pericyte-to-myofibroblast transition. scRNA-seq also identified the unexpected upregulation of nerve growth factor (NGF), which we confirmed in two mouse kidney fibrosis models. The Ngf receptor Ntrk1 is expressed in tubular epithelium in vivo, suggesting a novel interstitial-to-tubule paracrine signaling axis. Thus, KGli1 cells accurately model myofibroblast activation in vitro, and the development of this cell line provides a new tool to study resident mesenchymal stem cell-like progenitors in health and disease.

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Gene expression signature of the three major epidermal differentiation compartments: interfollicular epidermis (IFE), hair follicle (HF) and sebaceous gland (SG).

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Project description:To investigate the influecne of BMP signaling on Gli1+ cells, RNA-seq were performed after Smad4 or Alk3 knockout. Next, RNA-seq of Muc2-mCherry cells were performed to reveal the regulation of Gli1+ cell on intestinal epithelium. Finally, RNA-seq of colonic organoids were performed to reveal the regulation of IL-1 or IL-17 on epithelium. To investigate the influecne of BMP signaling on Gli1+ cells, RNA-seq were performed after Smad4 or Alk3 knockout. Next, RNA-seq of Muc2-mCherry cells were performed to reveal the regulation of Gli1+ cell on intestinal epithelium. Finally, RNA-seq of colonic organoids were performed to reveal the regulation of IL-1 or IL-17 on epithelium.

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The influence on Muc2 positive cells and Gli1 positive cells after the knockout of Smad4 or Alk3 in Gli1 positive cell and the effect of IL1 or IL17 on colonic organoids

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Expression profile of Gli1-expressing keratinocytes from mouse skin

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