RNA-seq of intestinal crypts vs intestinal smooth muscle with a specific MMP17 deletion compared to wild-type
Ontology highlight
ABSTRACT: We wanted to assess the role of a specific smooth muscle protein (MMP17) in two different intestinal compartments, the epithelium (crypts) and the smooth muscle. To do that we isolate intestinal crypts from wild-type (WT) and knockout (KO, Mmp17-/-) mice, and obtained clean strips of smooth muscle. After muscle dissociation, we obtained RNA directly from crypts and muscle, and it was used for RNA-seq. By comparing WT and KO samples we observed a higher impact in gene expression affecting crypts, even though MMP17 is only expressed in muscle. This helped us to identify altered signaling pathways in KO crypts that linked MMP17 with SMAD4 and BMP signaling.
Project description:To assess the role of LSD1 in mouse small intestinal epithelium, we isolated small intestinal crypts and villus from wild type (WT) (Villin-Cre -; Lsd1f/f) and intestinal-epithelial-specific knock-out (cKO) (Villin-Cre+; Lsd1f/f) mice. This experiment uses a new Cre strain with 100% deletion efficiency. RNA was directly isolated from intestinal crypt and villus, and this was used for RNAseq. Gene expression analysis of cKO derived crypt and villus provides a spatially restricted outlook on the maturation status of the intestinal epithelium in the villi and the absence of Paneth cells in the crypt.
Project description:In order to unravel the impact of intestinal smooth muscle tissue on the intestinal epithelium, we isolated clean smooth muscle, cultured it for 24h in DMEM-F12, and collected the supernatant (muscle-SN). This supernatant was used to treat small intestinal organoids (made of intestinal epithelium), compared to normal ENR treatment. After 5 days of muscle-SN exposure, we disrupted the organoids, and directly isolate the RNA. RNA-seq was performed in this sample to assess the genetic changes induced by muscle products.
Project description:To assess the role of LSD1 in mouse small intestinal epithelium, we isolated small intestinal crypts and villus from wild type (WT) (Villin-Cre -; Lsd1f/f) and intestinal-epithelial-specific knock-out (cKO) (Villin-Cre+; Lsd1f/f) mice. This experiment uses a new Cre strain with 100% recombination efficiency. RNA was directly isolated from the crypt and villus, and this was used for RNAseq. Gene expression analysis of cKO derived crypt and villus provides a spatially restricted outlook on the maturation status of the intestinal epithelium in the villi and the absence of Paneth cells in the crypt. Additionally, these mice were treated with antibiotics to study epithelium intrinsic changes related to LSD1 deletion but independent of the bacterial microbiome.
Project description:Polycomb-mediated gene repression plays an important role in adult stem cell maintenance. We knocked out (using the inducible AhCre-LoxP system) Polycomb genes Eed and Ezh2 in the intestine for 6 weeks, after which crypts - the small intestinal stem cell zone - were harvested and RNA sequenced. We found Wnt, Notch and cell cycle pathways to be affected in Eed knockout (KO) but not Ezh2 KO crypts. Direct targets of Eed were determined by comparing this data with ChIP-sequencing. Small intestinal crypt mRNA profiles of 6 weeks-induced 12 weeks old Eed KO, Ezh2 KO and WT mice (all triplicates) as well as 10 days-induced Eed KO and WT organoids (duplicates) were generated by RNA sequencing over two runs and using IlluminaHiseq2000 and Hiseq2500.
Project description:Polycomb-mediated gene repression plays an important role in adult stem cell maintenance. Direct targets of the Polycomb repressive complex PRC2 in th intestinal epithelium were revealed by performing ChIP-sequencing on crypt samples isolated from wild type murine small intestines. The resulting list of H3K27me3-enriched genes were compared with RNA-sequencing data from wild type and Eed knockout crypts. Crypts were isolated from wild type murine intestinal epithelium and subjected to ChIP using anti-H3K27me3 and anti-H3K27Ac antibodies, after which DNA isolated from extracted immunocomplexes was sequenced.
Project description:Cancer is characterized by gene expression aberrations. Studies have largely focused on coding sequences and promoters, despite the fact that distal regulatory elements play a central role in controlling transcription patterns. Here we utilize the histone mark H3K4me1 to analyze gain and loss of enhancer activity genome wide in primary colon cancer lines relative to normal colon crypts. We identified thousands of variant enhancer loci (VELs) that comprise a signature that is robustly predictive of the in vivo colon cancer transcriptome. Furthermore, VELs are enriched in haplotype blocks containing colon cancer genetic risk variants, implicating these genomic regions in colon cancer pathogenesis. We propose that reproducible changes in the epigenome at enhancer elements drive a unique transcriptional program to promote colon carcinogenesis. We used gene expression profiles to assess correlations between the colon cancer epigenome and transcriptional activity. RNA from normal colon epithelial crypt controls and primary colon cancer cell lines was hybridized to Affymetrix All Exon Human ST 1.0 microarrays.
Project description:Differentiation and specialisation of epithelial cells in the small intestine is regulated in two ways. First, there is differentiation along the crypt-villus axis of the intestinal stem cells into absorptive enterocytes, Paneth, goblet, tuft, enteroendocrine or M-cells, which is mainly regulated by WNT. Second, there is specialization along the cephalocaudal axis with different absorptive and digestive functions in duodenum, jejunum and ileum that is controlled by several transcription factors such as GATA4. However, so far it is unknown whether location-specific functional properties are intrinsically programmed within stem cells or if continuous signalling from mesenchymal cells is necessary to maintain the location-specific identity of the small intestine. By using the pure epithelial organoid technique, we show that region-specific gene expression profiles are conserved throughout long-term cultures of both mouse and human intestinal stem cells and correlated with differential Gata4 expression. Furthermore, the human organoid culture system demonstrates that Gata4-regulated gene expression is only allowed in absence of WNT signalling. These data show that location-specific function is intrinsically programmed in the adult stem cells of the small intestine and that their differentiation fate is independent of location-specific extracellular signals. In light of the potential future clinical application of small intestine-derived organoids, our data imply that it is important to generate GATA4-positive and GATA4-negative cultures to regenerate all essential functions of the small intestine. RNA sequencing of intestinal crypts, villi and cultured organoids derived from mouse duodenum, jejunum and ileum
Project description:Paneth cells are antimicrobial peptide-secreting cells located at the base of the crypts of the small intestine. The proteome of Paneth cells is not well defined because of their co-existence with stem cells making it difficult to culture Panth cells alone in vitro. Using a simplied toluidine blue O method for staining mouse intestinal tissue, laser capture microdissection (LCM) to isolate cells from the crypt region and surfactant assisted one pot protein digestion, we identified more than 1,300 proteins from crypts equivalent to 18,000 cells. Compared with the proteomes of villi and smooth muscle regions, the crypt proteome is highly enriched in defensins, lysozymes and other antimicrobial peptides that are characteristic of Paneth cells. The sensitivity of the LCM-based proteomics approach was also assessed using a smaller number of cell equivalent tissues, a comparable proteomic coverage can be achieved with 3,600 cells. This work is the first proteomics study of intestinal tissue enriched with Paneth cells. The simplied workflow enables profiling of Paneth cell associated pathological changes at the proteome level directly from frozen intestinal tissue. It may also be useful for proteomics studies of other spatially resolved cell types from other tissues.
Project description:The goal of this study was to identify genes that are differentially expressed during a 24 hour Notch blockade and whether a Wnt antagonist (anti-Lrp6) could block this effect. 3 condition experiment, Control untreated compared to N1N2 treated, and N1N2 treated compared to N1N2_Lrp6 treated. 5 biological replicates per condition.
Project description:The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of approximately six cycling Lgr5+ stem cells at the bottoms of small intestinal crypts1. We have now established long-term culture conditions under which single crypts undergo multiple crypt fission events, whilst simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5+ stem cells can also initiate these crypt-villus organoids. Tracing experiments indicate that the Lgr5+ stem cell hierarchy is maintained in organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche. Keywords: expression profiling Freshly isolated small intestinal crypts from two mice were divided into two parts. RNA was directly isolated from one part (RNeasy Mini Kit, Qiagen), the other part was cultured for one week according to the conditions described in the associated paper, followed by RNA isolation. We prepared labeled cRNA following the manufacturer’s instruction (Agilent Technologies). Differentially labelled cRNA from small intestinal crypts and organoids were hybridised separately for the two mice on a 4X44k Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in two dye swap experiments, resulting in four individual arrays.