Effect of depletion of FABP4 in mice intestinal epithelial cells on gene expression
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE200967 | GEO | 2022/04/25
REPOSITORIES: GEO
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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.
2022-10-12 | PXD034143 | Pride
Project description:At the base of the intestinal crypt, long-lived Lgr5+ stem cells are intercalated by Paneth cells that provide essential niche signals for stem-cell maintenance. This unique epithelial anatomy makes the intestinal crypt one of the most accessible models for the study of adult stem cell biology. The glycosylation patterns of this compartment are poorly characterized and the impact of glycans on stem cell differentiation remains largely unexplored. We found that Paneth cells, but not Lgr5+ stem cells, express abundant terminal N-acetyllactosamine (LacNAc). Employing an enzymatic method to edit glycans in cultured crypt organoids, we assessed the functional role of LacNAc in the intestinal crypt. We show that blocking access to LacNAc on Paneth cells leads to hyperproliferation of the neighbouring Lgr5+ stem cells, which is accompanied by the down-regulation of genes that are known as negative regulators of proliferation
2017-12-27 | GSE108512 | GEO
Project description:Nod2 has been extensively characterized as a bacterial sensor that induces an antimicrobial and inflammatory gene expression program. Therefore, it is unclear why Nod2 mutations that disrupt bacterial recognition are paradoxically among the highest risk factors for Crohn’s disease, which involves an exaggerated immune response directed at intestinal bacteria. Previous studies from our lab have shown that mice deficient in Atg16L1, another Crohns disease susceptibility gene, develop abnormalities in Paneth cells, specialized epithelial cells in the small intestine involved in antimicrobial responses. The goal of our study is to determine whether Nod2 deficiency leads to differences in the transcriptional profile of Paneth cells, ultimately leading to small intestinal inflammation. Small intestinal sections (ileum) of 8 week old WT and Nod2-/- mice were fixed in methacarn and embedded in paraffin. The Leica LMD6000 Laser Microdissection system was used to capture crypt base epithelial cells to enrich for Paneth cells. RNA was extracted from these cells, followed by cDNA synthesis and qPCR to confirm enrichment of Paneth cells using unique markers (a-defensins).
2014-07-01 | E-GEOD-58948 | biostudies-arrayexpress
Project description:Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., {alpha}-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens. Keywords: other
2005-07-18 | GSE2958 | GEO
Project description:Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., {alpha}-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens. Total RNA was extracted from pooled small intestines of three WT and three CF mice using Tri-Reagent (Molecular Research Center, Cincinnati, OH), and poly(A) RNA was purified by using the MicroPoly(A) mRNA purification kit (Ambion, Austin, TX). The WT and CF poly(A) RNA samples were sent to IncyteGenomics (St. Louis, MO) where they were labeled with cyanine 3 (Cy3) and Cy5, respectively, and hybridized with the UniGEM1.31 array representing 9,570 known genes and expressed sequence tags.
2010-06-09 | E-GEOD-2958 | biostudies-arrayexpress
Project description:Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, small cycling cells located at crypt bottoms1, 2. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells, that are known to produce bactericidal products such as lysozyme and cryptdins/defensins3. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells4. Here, we note a close physical association of Lgr5 stem cells with Paneth cells in vivo and in vitro. CD24+ Paneth cells express EGF, TGF?, Wnt3 and the Notch-ligand Dll4, all essential signals for stem cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell. We used intestinal cell fractions from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. RNA was isolated from two FACS sorted cell populations: stem cells were sorted based on high level of GFP expression (GFPhi) and Paneth cells were sorted based on high level of CD24 expression (CD24hi) and high side-scatter (SSChi). Differentially labelled cRNA from GFPhi and CD24hi/SSChi cells from two different sorts (each combining ten individual mice) were hybridized on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in two dye swap experiments, resulting in four individual arrays.
2010-11-29 | E-GEOD-25109 | biostudies-arrayexpress
Project description:Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, small cycling cells located at crypt bottoms1, 2. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells, that are known to produce bactericidal products such as lysozyme and cryptdins/defensins3. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells4. Here, we note a close physical association of Lgr5 stem cells with Paneth cells in vivo and in vitro. CD24+ Paneth cells express EGF, TGFα, Wnt3 and the Notch-ligand Dll4, all essential signals for stem cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.
2010-11-29 | GSE25109 | GEO
Project description:To assess the role of LSD1 in human intestinal epithelium, human small intestinal organoids were treated with an inhibitor for LSD1 (GSK-LSD1) and compared to untreated organoids. The organoids were grown with specific conditions where Paneth cells are present in the organoids as similar experiments in mice show that Paneth cells disappear upon GSK-LSD1 treatment. Similar to mouse intestinal organoids, Paneth cells dissappear upon GSK-LSD1 treatment. Furthermore, we used these gene set enrichment analysis on these microarray data to show that these human intestinal organoids have a similar phenotype as mice epithelium without LSD1.
2020-11-16 | E-MTAB-7863 | biostudies-arrayexpress
Project description:Acetylcholine (ACh) has been considered a neurotransmitter residing in central, parasympathetic and neuromuscular synapses of mammals. Here, experiments using crypt-villus organoids that lack nerve and immune cells in culture led us to suggest that endogenous ACh is synthesized in the intestinal epithelium to evoke growth and differentiation of the organoids through activation of muscarinic ACh receptors (mAChRs). The extracts of the cultured organoids exhibit a noticeable capacity for ACh synthesis that is sensitive to a potent inhibitor of choline acetyltransferase (ChAT). Imaging mass spectrometry reveals distribution of endogenous ACh that is localized in intestinal epithelial layer in the cultured organoids as well as in mouse small intestinal epithelium in vivo, suggesting non-neural resources of ACh. Treatment of organoids with carbachol down-regulates growth of organoids and expression of marker gene for each epithelial cell. On the other hand, antagonists for mAChRs enhances growth and differentiation of organoids, indicating involvement of mAChRs in regulating proliferation and differentiation of Lgr5-positive stem cells. Collectively, our data provide evidence that endogenous ACh released from intestinal epithelium maintains homeostasis of intestinal epithelial cell growth and differentiation via mAChRs in mice. Gene expression patters of gut, crypt, y-organoid and o-organoid, respectively
2013-08-15 | E-GEOD-49864 | biostudies-arrayexpress
Project description:To compare miRNA expression profiles between freshly isolated intestinal epithelial cells and cultured organoids in mice. Intestinal organoids largely resembled intestinal epithelial cells in their miRNA profiles. Although the expression levels of some miRNAs were different between crypt and villus epithelial cells, such expression patterns were not reproduced during the maturation of intestinal organoids.
2018-05-25 | GSE99237 | GEO