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ZIP7 is indispensable for proliferation of intestinal epithelial cells and maintenance of intestinal stem cells

ABSTRACT: Intestinal epithelium are generated by intestinal stem cells, which are recognized morphologically as slender columnar cells at the base of the crypt. Stem cells produce transit-amplifying (TA) cells, which divide a number of times and the daughter cells differentiate into absorptive enterocytes as well as secretory-lineages. Intestinal stem cells highly express Lgr5 which is decreased in TA cells. Here, we show that the zinc transported SLC39A7/ZIP7 is essential for the proliferation of TA cells and maintenance of intestinal stem cells. Lgr5Med TA cells derived from Zip7-deficient mice upregulated the expression of unfold protein responses-related genes including pro-apoptotic genes, indicating of induction of ER stress in these cells. The same effect was seen in Lgr5Hi stem cells derived from Zip7-deficient mice. We conclude that ZIP7 is fundamental to the maintenance of crypt homeostasis by resolving ER stress. Small intestinal crypts were isolated form tamoxifen-treated control (Zip7flox/+, Villin-CreERT2, Lgr5-EGFP-ires-CreERT2) and tamoxifen-treated Zip7â??IEC (Zip7flox/flox, Villin-CreERT2, Lgr5-EGFP-ires-CreERT2) mice. We FACS purified intestinal crypt cells according to their Lgr5 expression levels. RNA was isolated from four FACS sorted cell populations: Lgr5Hi cells and Lgr5Med cells derived from control mice, Lgr5Hi cells and Lgr5Med cells derived from Zip7â??IEC mice. Isolated RNA was analyzed using the Affymetrix platform.

ORGANISM(S): Mus musculus

SUBMITTER: Yoshiki Mochizuki

PROVIDER: E-GEOD-72190 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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ZIP7 is indispensable for proliferation of intestinal epithelial cells and maintenance of intestinal stem cells

Project description:Intestinal epithelium are generated by intestinal stem cells, which are recognized morphologically as slender columnar cells at the base of the crypt. Stem cells produce transit-amplifying (TA) cells, which divide a number of times and the daughter cells differentiate into absorptive enterocytes as well as secretory-lineages. Intestinal stem cells highly express Lgr5 which is decreased in TA cells. Here, we show that the zinc transported SLC39A7/ZIP7 is essential for the proliferation of TA cells and maintenance of intestinal stem cells. Lgr5Med TA cells derived from Zip7-deficient mice upregulated the expression of unfold protein responses-related genes including pro-apoptotic genes, indicating of induction of ER stress in these cells. The same effect was seen in Lgr5Hi stem cells derived from Zip7-deficient mice. We conclude that ZIP7 is fundamental to the maintenance of crypt homeostasis by resolving ER stress.

2016-08-19 | GSE72190 | GEO

Gene expression profiling along the intestinal crypt axis.

Project description:The identification of Lgr5 as an intestinal stem cell marker has made it possible to isolate and study primary intestinal stem cells. Transcriptional differences between intestinal stem cells can be explored by the use of the Lgr5-eGFP-ires-CreERT2 knock-in mouse. In this mouse model GFP expression is driven from the Lgr5 locus, leading to highest GFP levels in the Lgr5 positive cells. Yet, due to the stability of the GFP protein, it is distributed upon cell division to the daughter cells. Here we arbitrarily sorted crypt cells based on GFP expression into five fractions. The fraction with the lowest GFP level is named 1+, the fraction with the highest level 5+. We used cell fractions of intestines from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. RNA was isolated from five FACS sorted cell populations, with fraction 5+ expressing GFP at highest levels and 1+ expressing GFP at lowest levels. Differentially labelled cRNA from fractions 1+ to 4+ were hybridized against fraction 5+ on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in dye swap experiments, resulting in eight individual arrays.

2012-08-15 | E-GEOD-36497 | biostudies-arrayexpress

Gene expression signature of fast and slow cycling intestinal crypt base columnar cell populations

Project description:The identification of Lgr5 as an intestinal stem cell marker has made it possible to isolate and study primary stem cells from small intestine. Using the cell cycle specific expression og the mKi67 gene, we generated a novel Ki67-RFP knock-in allele which identifies dividing cells. Using Lgr5-GFP;Ki67-RFP mice, we isolated CBCs with distinct Wnt signaling levels and cell cycle features, and analyzed their global gene expression pattern using microarrays. We concluded that the cycling Lgr5hi stem cells exit the cell cycle in transition into the secretory lineage. Lgr5med Ki67low intermediate precursors reside in the zone of differentiation, resemble quiescent stem cells and generate the Dll1+ secretory precursors and the label retaining cells. Our findings support the cycling stem cell hypothesis and highlight the heterogeneity of early progenitors during lineage commitment. We used cell fractions of intestines from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter, and Ki67-TagRFP mice where the RFP is fused to the C-terminus of the endogenous Ki67 gene. RNA was isolated from several FACS sorted cell populations of combinations expressing different levels of GFP and RFP: GFP high RFP high (Lgr5hi Ki67hi), GFP high RFP low (Lgr5hi Ki67low), GFP medium RFP high (Lgr5med Ki67high) and GFP medium RFP low (Lgr5med Ki67low). Purified RNA was processed, hybridized, and scanned according to the manufacturerM-bM-^@M-^Ys protocol and were hybridized on Affymetrix Mouse Gene ST 1.1 arrays).

2014-09-08 | E-GEOD-52813 | biostudies-arrayexpress

Murine small intestinal crypt cell line IEC-6: control vs. TGF-beta stimulated

2012-06-19 | GSE20982 | GEO

Gene expression profiling along the intestinal crypt axis.

2012-08-15 | GSE36497 | GEO

Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts

Project description:Wnt/b-catenin signaling supports intestinal homeostasis by regulating proliferation in the crypt. Multiple Wnts are expressed in Paneth as well as other intestinal epithelial and stromal cells. Ex vivo, Wnts secreted by Paneth cells can support intestinal stem cells when Wnt signaling is enhanced with supplemental R-Spondin 1 (RSPO1). However, in vivo, the source of Wnts in the stem cell niche is less clear. Genetic ablation of Porcn, an endoplasmic reticulum resident O-acyltransferase that is essential for the secretion and activity of all vertebrate Wnts, confirmed the role of intestinal epithelial Wnts in ex vivo culture. Unexpectedly, mice lacking epithelial Wnt activity (PorcnDel/Villin-Cre mice) had normal intestinal proliferation and differentiation, as well as successful regeneration after radiation injury, indicating epithelial Wnts are dispensable for these processes. Consistent with a key role for stroma in the crypt niche, intestinal stromal cells endogenously expressing Wnts and Rspo3 support the growth of PorcnDel organoids ex vivo without RSPO1 supplementation. Conversely, increasing pharmacologic PORCN inhibition, affecting both stroma and epithelium, reduced Lgr5 intestinal stem cells, inhibited recovery from radiation injury, and at the highest dose fully blocked intestinal proliferation. We conclude that epithelial Wnts are dispensable, and that stromal production of Wnts can fully support normal murine intestinal homeostasis. Microarray was performed on samples enriched for stromal or epithelial cells from small intestine from Porcn(Del)/Villin-Cre and Porcn(WT)/Villin-Cre male C57Bl/6 mice.

2014-04-19 | E-GEOD-56911 | biostudies-arrayexpress

Murine small intestinal crypt cell line IEC-6: control vs. TGF-beta stimulated

Project description:microRNA profiling of rat small intestinal crypt cell IEC-6. Comparing control untreated with cells treated with transforming growth factor-beta (TGF-beta). TGF-beta stimulated cell differentiation, as observed in the stimulation of intestinal epithelial cell markers (alkaline phophotase, villin, aminopeptidase N, etc.). Two condition experiment. Control vs TGF-beta treatment. Biological replicates: 3 control, 3 treated. Independently grown and harvested. One replicate per array

2012-06-18 | E-GEOD-20982 | biostudies-arrayexpress

Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts

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

Transcription profiling of mouse intestinal cells expressing Lgr5 at high or low levels or with Ascl2 conditionally knocked out shows Ascl2 controls intestinal stem cell fate

Project description:The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these so called Crypt Base Columnar (CBC) cells. One of the genes within this stem cell signature is the Wnt target Ascl2. Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and de novo crypt formation on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the CBC stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate. Experiment Overall Design: For the stem cell signature we used cell fractions of intestines from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. RNA was isolated from two FACS sorted cell populations, one expressing GFP at high levels (GFPhi) and the other expressing GFP at low levels (GFPlo). For the analysis of Ascl2 target genes RNA was isolated from intestinal epithelial cells of Ah-Cre/Ascl2floxed/floxed animals and Ah-Cre/Ascl2floxed/wt control animals 3 and 5 days post induction. Differentially labelled cRNA from GFPhi and GFPlo cells from two different sorts (each combining three different mice) were hybridised on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in two dye swap experiments, resulting in four individual arrays. For the Ascl2 target gene analysis we analyzed the 3 and 5 days PI experiments in two dye swap experiments, resulting in four individual arrays.

2009-03-25 | E-GEOD-14201 | biostudies-arrayexpress

RNA-seq of mouse Lgr5+ intestinal stem cells and progenitors following Lkb1 deletion against controls

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

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