Project description:Goal of this study was to examine gene expression changes upon conditional deletion of beta-catenin at E10.5 in dermal fibroblasts. Method: Skin tissues were treated in 0/25% trypsin for 15 min at 37 degrees C to obtain single cells for FACS sorting and collected in RNA later. RNA was extracted with Karcturus pico pure kit. The RT and amplification was done using Nugen's ovation RNA-seq system V2. Libraries for sequencing were prepared with Illumina TruSeq kit. Transcriptome of FACS sorted E13.5 Engrailed1; RRYFP lineage-marked dorsal dermal fibroblasts was generated next-gen sequencing, in triplicate, using Illumina HiSeq machine. The sequence reads that passed quality filters were analyzed by TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: We mapped about 33-49 million sequence reads per sample and obtained 76-79% of uniquely mapped percentage. We assembled the reads to the mouse ref genome (build mm10).
Project description:Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced (EF skin) by transgenic epidermal activation of beta-catenin. We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of beta-catenin signalling. In contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal beta-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis. We have isolated the following populations of cells from mouse back skin by flow cytometry: 1A) GFP+ WT neonatal dermal fibroblasts, 1B) ItgA6+ WT neonatal epidermal keratinocytes, 2A) GFP+ WT telogen dermal fibroblasts, 2B) ItgA6+ WT telogen epidermal keratinocytes, 3A) GFP+ D2 transient activation (anagen) dermal fibroblasts, 3B) ItgA6+ D2 transient activation (anagen) epidermal keratinocytes, 4A) GFP+ D2 sustained activation (ectopic follicles) dermal fibroblasts, 4B) ItgA6+ D2 sustained activation (ectopic follicles) epidermal keratinocytes