Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE27904 | GEO | 2012/03/10
SECONDARY ACCESSION(S): PRJNA138217
REPOSITORIES: GEO
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Project description:Gene inactivation of the orphan G protein-coupled receptor Lgr4, a paralog of the epithelial stem cell marker Lgr5, results in 50% decrease of epithelial cell proliferation and 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, Lgr4-deficient crypts or progenitors, but not Lgr5-deficient progenitors, die rapidly with dramatic downregulation of stem cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by LiCl addition to the culture medium, but not by Wnt agonists. Our results identify Lgr4 as a permissive factor of the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy. Microarray hybridization was performed on LGR4 KO intestinal crypts at day 0, day 0.5 and day 1 versus wild-type crypts. The effects of LiCl treatment on LGR4 KO crypts at day1 versus control cells were investigated. After amplification and labelling, sample pairs were hybridized onto Mouse Exonic Evidence Based Oligonucleotide (MEEBO) arrays containing on average 38784 mouse 70mer oligonucleotide probes (Stanford University, US). Hybridizations were replicated with dye swap.
2012-03-10 | E-GEOD-27904 | biostudies-arrayexpress
Project description:The adult stem cell marker Lgr5 and its close relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. Conditional deletion of the two genes in the gut impairs expression of Wnt target genes and results in rapid demise of intestinal crypts, thus phenocopying the effects of Wnt pathway inhibition. By mass-spectrometry, we find that Lgr4 and Lgr5 associate with the endogenous Wnt co-receptors Frizzled-5/7 and Lrp5/6 in HEK293 cells and in colorectal cancer cells. We also find that soluble Rspondin1, a Wnt pathway agonist, specifically binds to endogenous Lgr4 on HEK293 cells. In these cells, soluble Rspondin1 potently enhances canonical Wnt signals initiated by Wnt3A. Removal of Lgr4 does not affect Wnt3A-induced signals, but abrogates the Rspondin1-mediated enhancement of these signals, a phenomenon rescued by re-expression of Lgr4, -5 or -6. Rspondin1 and can be rescued by Wnt pathway activation. Lgr4/5/6 are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble Rspondin proteins.
2011-07-04 | GSE28265 | GEO
Project description:The adult stem cell marker Lgr5 and its close relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. Conditional deletion of the two genes in the gut impairs expression of Wnt target genes and results in rapid demise of intestinal crypts, thus phenocopying the effects of Wnt pathway inhibition. By mass-spectrometry, we find that Lgr4 and Lgr5 associate with the endogenous Wnt co-receptors Frizzled-5/7 and Lrp5/6 in HEK293 cells and in colorectal cancer cells. We also find that soluble Rspondin1, a Wnt pathway agonist, specifically binds to endogenous Lgr4 on HEK293 cells. In these cells, soluble Rspondin1 potently enhances canonical Wnt signals initiated by Wnt3A. Removal of Lgr4 does not affect Wnt3A-induced signals, but abrogates the Rspondin1-mediated enhancement of these signals, a phenomenon rescued by re-expression of Lgr4, -5 or -6. Rspondin1 and can be rescued by Wnt pathway activation. Lgr4/5/6 are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble Rspondin proteins. We used two different experimental setups to show that concomitant deletion of Lgr4 and Lgr5 affects the expression of Wnt pathway target genes. First, we determined the downregulated genes after Lgr4/5 deletion in AhCre_Lgr4fl/fl_Lgr5 fl/fl mice on day 1 post-deletion, before any histological changes are apparent in these mice (Lgr4/5 gene signature). Next, we acutely withheld the Wnt agonist Rspondin1 from in vitro crypt organoid cultures and performed differential gene expression before, and 1 day after withdrawal (Rspondin1 withdrawal signature). For better comparison we also deleted the Lgr4 and Lgr5 in vitro in organoids isolated from VillinCre_Lgr4fl/fl_Lgr5 fl/fl mice, and performed microarrays 1 day after deletion in vitro. Furthermore, we used published expression data from Apcfl/fl mice (Apc deletion signature). Deletion of Apc results in the immediate upregulation of Wnt pathway target genes. Comparison of the Lgr4/5 gene signature to both the Rspondin1 withdrawal signature and the Apc deletion signatures using Gene Set Enrichment Analysis (GSEA) showed that the genes deregulated after simultaneous deletion of Lgr4 and -5 are in the majority under the control of the Wnt pathway.
2011-07-04 | E-GEOD-28265 | 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:Paneth cells (PCs) are long-lived secretory cells that reside at the bottoms of small intestinal crypts. Besides serving as niche cells for the neighboring Lgr5-positive stem cells, PCs secrete granules containing a broad spectrum of antimicrobial proteins, including lysozymes and defensins1. Here, we have used single-cell RNA sequencing to explore PC differentiation. We found a maturation gradient from early secretory progenitors to mature PCs, capturing the full maturation path of PCs. Moreover, differential expression of a subset of defensin genes in lysozyme-high PCs, e.g. Defa20, reveals at least two distinct stages of maturation. We traced Lgr5+ stem cells from Lgr5-CreERT2 C57Bl6/J mice bred to a Rosa26LSL-YFP reporter mice and sorted YFP+ cells 5 days, 3 weeks and 8 weeks after tamoxifen injection.
2016-06-23 | E-GEOD-76408 | 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:Perturbed intestinal epithelial homeostasis demonstrated as decreased Lgr5+ intestinal stem cells (Lgr5 ISCs) and increased secretory lineages were observed in our study where Lkb1 was specfically deleted in Lgr5 ISCs using Lgr5-EGFP-creERT2 (Tamoxifen) deletor. To gain mechanistic insight how Lkb1 maintains intestinal epithelial stem cell homeostasis, Lkb1 deficient ISCs (Lgr5-high cells) and progenitors (Lgr5-low cells) are isolated by flow cytometry and profiled by RNA sequencing to compare with controls (Lkb1 wild type ISCs and progenitors).
2020-02-29 | E-MTAB-6501 | biostudies-arrayexpress
Project description:Paneth cells (PCs) are long-lived secretory cells that reside at the bottoms of small intestinal crypts. Besides serving as niche cells for the neighboring Lgr5-positive stem cells, PCs secrete granules containing a broad spectrum of antimicrobial proteins, including lysozymes and defensins1. Here, we have used single-cell RNA sequencing to explore PC differentiation. We found a maturation gradient from early secretory progenitors to mature PCs, capturing the full maturation path of PCs. Moreover, differential expression of a subset of defensin genes in lysozyme-high PCs, e.g. Defa20, reveals at least two distinct stages of maturation.
2016-06-23 | GSE76408 | GEO
Project description:The goal of this project is to generate transcriptome profiling of Lgr5-GFP high intestinal stem cells (High)/Lgr5-GFP low progenitors (Low) and CD24+C-kit+ Paneth (PA) cells for a systemic analysis of cellular pathways involved in responses to fasting and aging.
2019-07-19 | GSE122230 | GEO
Project description:Inhibition of leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) prevents the development of Mixed Lineage Leukemia (MLL)-arranged AML in mice and suppresses activation of Wnt/beta-catenin signaling. To identify key downstream targets of Lgr4, we performed genome-wide gene expression analysis. Our microarray analysis and subsequent in vivo studies demonstrate a novel functional role for growth arrest and DNA damage-inducible 45 (Gadd45a) in promoting in vivo self-renewal of leukemic stem cells in MLL-rearranged AML. GFP-positive leukemic cells flow-sorted by BD Influx cell sorter from bone marrow of primary AML mice induced by MLL-AF9 oncogene were lentivirally transduced with Lgr4 shRNA (MSH040504-3-LVRU6MP, GeneCopoeia) or Scrambled control (CSHCTR001-LVRU6MP, GeneCopoeia), and replated in methylcellulose supplemented with IL3. Each group contains triplicate samples.
2023-08-30 | E-MTAB-4773 | biostudies-arrayexpress