Project description:The purpose of this study is to compare the cellular populations present within granuloma (Sca1+) and non-granuloma (Sca-1–) crypt epithelum arising from parasitic H polygyrus infections.
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
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.
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: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:We used the mouse pathogen Citrobacter rodentium to model gut infections. Following oral inoculation C. rodentium resides in the caecum for the first 3 days, before it infects the colon on the 4th day. Here we show that while the host is unresponsive to the infection on day 3, there is an abrupt reprogramming of the cellular composition of the crypt, involving depletion of goblet and deep crypt secretory cells, as well as metabolism (e.g. simultaneous up-regulation of cholesterol biogenesis, import and efflux), DNA damage repair and proliferation, which correlated with Ki67 staining, on day 4. Reduction in the abundance of proteins involved in the TCA cycle and oxidative phosphorylation, leading to oxygenation of the gut, coincided with instant expansion of mucosal-associated Enterobacteriaceae. These results show that sensing a small number of pathogenic bacteria triggers immediate intrinsic changes to the epithelium physiology and the microbiota, which parallel innate gut immune responses.
Project description:To assess the role of LSD1 in mouse small intestinal epithelium, we isolated small intestinal crypts from wild type (WT) (Villin-Cre -; Lsd1f/f) and intestinal-epithelial-specific knock-out (KO) (Villin-Cre+; Lsd1f/f) mice. We dissociated crypts into single cells, and FACS sorted Epcam+ cells, to avoid immune-cell contamination. RNA was directly isolated from these sorted cells, and this was used for RNA seq. As KO crypts are different from WT crypts (KO crypts lack Paneth cells), identifying genes specifically regulated by LSD1 helps us to identify how LSD1 regulates intestinal crypt biology. Specifically, because we were able to combine this with ChIP-seq of the same cells, to identify where H3K4me1 levels (target of the histone demethylase LSD1) were different in the genome.
Project description:We obtained a spatial measurement of RNA and Proteins in the small intestinal epithelium along the crypt-villus axis. We found that both were spatially heterogeneous, yet often spatially anti-correlated. We developed a Bayesian approach to infer protein translation and degradation rates from the combined spatial profiles, and demonstrate that space-independent protein-synthesis delays can explain the mRNA-protein discordances. Our work provides a proteomic spatial blueprint of the Intestinal epithelium and highlights the importance of protein measurements for inferring states of tissue cells that operate outside of steady state