Gene expression in intestinal stem cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
SUBMITTER: Juerg Straubhaar
PROVIDER: E-GEOD-58351 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Gene expression profiling identifies the zinc-finger protein Charlatan as a regulator of intestinal stem cells in Drosophila.
Amcheslavsky Alla A Nie Yingchao Y Li Qi Q He Feng F Tsuda Leo L Markstein Michele M Ip Y Tony YT
Development (Cambridge, England) 20140701 13
Intestinal stem cells (ISCs) in the adult Drosophila midgut can respond to tissue damage and support repair. We used genetic manipulation to increase the number of ISC-like cells in the adult midgut and performed gene expression profiling to identify potential ISC regulators. A detailed analysis of one of these potential regulators, the zinc-finger protein Charlatan, was carried out. MARCM clonal analysis and RNAi in precursor cells showed that loss of Chn function caused severe ISC division def ...[more]
Similar Datasets
Project description:Genetic Manipulation to increase number of ISC (intestinal stem cells) and gene expression profiling to identify ISC regulators
2014-06-16 | GSE58351 | GEO
Project description:Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation. Two samples of intestinal subepithelial myofibroblasts were used in this study.
2013-12-20 | E-GEOD-52402 | biostudies-arrayexpress
Project description:Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.
2013-12-20 | GSE52402 | GEO
Project description:Purpose: We isolated Drosophila midgut cells : Delta+ intestinal stem cells (ISCs), Su(H)+enteroblasts (EBs), Esg+ cells (ISC+EB), Myo1A+Enterocytes (ECs), Pros+Enteroendocrine cells (EEs) and How+Visceral muscle cells (VM) from whole midguts to identify stem cell specific genes and study cell type specificities of midgut cells. We also isolated all the cell types from the 5 major regions (R1-R5) of the Drosophila midgut to study differences in cells in different regions. Methods: 3-7 day old female flies were dissected. Flies with GFP/YFP marking different cell types (using the GAL4-UAS system) were used to separate cells of the midgut.The midguts were dissociated with Elastase and FACS sorted using FACS AriaIII. RNA was extracted, amplified and sequenced. Whole midgut samples were sequenced on Illumina GAIIX and regional cell populations were sequenced on HiSeq2000. Methods:Raw fastqc reads were mapped to the Drosophila genome (Drosophila_melanogaster.BDGP5.70.dna.toplevel.fa) using Tophat 2.0.9 at default (using boost_1_54_0, bowtie2-2.1.0, samtools-0.1.19). Methods: For differential expression analysis, DESeq (p-value adjustment 0.05 by method Benjamini-Hochberg) was used. The reads were normalized also to Reads per kilobase of transcript per million mapped reads (RPKM). Results: More than 50% of the genome is expressed in the adult midgut (FlyAtlas- Chintapalli et al., 2007), of these genes about 50% (2457) were differentially expressed (DE) between all 4 cell types (ISCs, EBs, ECs and EEs) atleast 2 folds with 95% confidence Results: 159 genes that were specifically enriched in ISCs, 509 genes were specifically repressed in ISCs Conclusions: Our study represents the first detailed analysis of Drosophila intestinal cell transcriptomes, with biologic replicates, generated by RNA-seq technology.Our data facilitates comparative investigations of expression profiles of cells and reveals novel stem cell genes. Further region specific profiling adds precision to the analysis of variances in the midgut regions. We identify transcriptional regulators and regional transcription factors which modulate the midgut physiology. The dataset will be a great resource for hypothesis generation, tool building and fine tuned studies on the Drosophila midgut. mRNA profiles of Drosophila intestinal cells from whole midguts and midgut regions were generated by Deep Sequencing. Whole midgut profiles were generated in triplicates (Illumina GAIIx, 72 bp read length) and regional cell type profiles were genrated in duplicates (HiSeq 2000, 50bp read length).
2015-05-23 | E-GEOD-61361 | biostudies-arrayexpress
Project description:Constitutive activation of the Wnt pathway leads to adenoma formation, an obligatory step towards intestinal cancer. In view of the established role of Wnt in regulating stemness, we attempted the isolation of cancer stem cells (CSCs) from Apc- and Apc/KRAS-mutant intestinal tumours. Whereas CSCs are present in malignant Apc/KRAS–mutant carcinomas, they appear to be very rare (<10-6) in the benign Apc–mutant adenomas. In contrast, the Lin-CD24hiCD29+ subpopulation of adenocarcinoma cells appear to be enriched in CSCs with increased levels of active -catenin. Expression profiling analysis of the CSC-enriched subpopulation confirmed their enhanced Wnt activity and revealed additional differential expression of other signalling pathways, growth factor binding proteins, and extracellular matrix components. As expected, genes characteristic of the Paneth cell lineage (e.g. defensins) are co-expressed together with stem cell genes (e.g. Lgr5) within the CSC-enriched subpopulation. This is of interest as it may indicate a cancer stem cell niche role for tumor-derived Paneth-like cells, similar to their role in supporting Lgr5+ stem cells in the normal intestinal crypt. Overall, our results indicate that oncogenic KRAS activation in Apc-driven tumours results in the expansion of the CSCs compartment by increasing b-catenin intracellular stabilization. To identify molecular differences between stem-like and more differentiated (bulk) tumour cells from Apc1638N/+ and Apc1638N/+/KRASV12G intestinal adenomas and adenocarcinomas, we isolated total RNA from 104 Lin-CD24hiCD29+, Lin-CD24medCD29+/Lin-CD24loCD29+ and Lin- (bulk) tumour cells from 5 individual mice of each genotype (Apc1638N/+ and Apc1638N/+/KRASV12G). Total RNA samples were then employed to hybridize oligonucleotide microarrays (Affymetrix Mouse Genome 430A 2.0 Array) according to conventional protocols.
2013-10-01 | E-GEOD-47772 | biostudies-arrayexpress
Project description:Background & Aims: Hierarchical organization of intestine relies on their stem cells by self-renew and producing committed progenitors. Although signals like Wnt are known to animate the continued renewal by maintaining intestinal stem cells (ISCs) activity, molecular mechanisms especially E3 ubiquitin ligases that modulate ISCs ‘stemness’ and supportive niche have not been well understood. Here, we investigated the role of Cullin 4B (Cul4b) in regulating ISC functions. Methods: We generated mice with intestinal epithelial-specific disruption of Cul4b (pVillin-cre; Cul4bfn/Y), inducible disruption of Cul4b (Lgr5-creERT2; Cul4bfn/Y, CAG-creERT2; Cul4bfn/Y) and their control (Cul4bfn/Y). Intestinal tissues were analyzed by histology, immunofluorescence, RNA sequencing and mass spectrum. Intestinal organoids deprived from mice with pVillin-Cre; Cul4bfn/Y, Lgr5-Cre; Cul4bfn/Y, Tg-Cul4b and their controls were used in assays to measure intestinal self-renewal, proliferation and differentiation. Wnt signaling and intestinal markers were analyzed by immunofluorescence and immunoblot assays. Differential proteins upon Cul4b ablation or Cul4b-interacting proteins were identified by mass spectrometry. Results: Cul4b specifically located at ISCs zone. Block of Cul4b impaired intestinal homeostasis maintenance by reduced self-renewal and proliferation. Transcriptome analysis revealed that Cul4b-null intestine lose ISC characterization and showed disturbed ISC niche. Mechanistically, reactivated Wnt pathway could recover intestinal dysfunction of Cul4b knockout mice. Analysis of differential total and ubiquitylated proteins uncovered the novel targeting substrate of Cullin-Ring ubiquitin ligase 4b (CRL4b), immunity-related GTPase family M member 1 (Irgm1) in intestine. Decreased Irgm1 rescued abnormally interferon signaling, overemphasized autophagy and downstream phosphate proteins in Cul4b knockout mice. Conclusion: We conclude that Cul4b is essential for ISC self-renewal and Paneth cell function by targeting Irgm1 and modulating Wnt signaling. Our results demonstrate that Cul4b is a novel ISC stemness and niche regulator.
2022-08-11 | PXD021528 | Pride
Project description:The gut and liver have been recognized to mutually communicate through the biliary tract, portal vein and systemic circulation, but it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy, transcriptomics and proteomics profiling, we identified pigment epithelium-derived factor (PEDF), as a liver-derived soluble Wnt inhibitor, that can restrain intestinal stem cells (ISC) hyperproliferation for gut homeostasis by competing with Wnt ligands and suppressing Wnt/beta-catenin signaling pathway. In turn, microbial danger signals from intestinal inflammation can be sensed by the liver to repress PEDF production via peroxisome proliferator-activated receptor-alpha (PPAR alpha), liberating ISC proliferation to accelerate tissue repair in the gut. Further, treatment of mice with fenofibrate, a clinical agent of PPARalpha agonist for hypolipidemia enhances the susceptibility of colitis via PEDF activity. Therefore, we identified a distinct role of PEDF to calibrate ISC expansion for intestinal homeostasis via reciprocal interactions between the gut and liver.
2023-03-03 | MSV000091409 | MassIVE
Project description:Abstract: RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine, and the mechanisms of how they contribute to tumorigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine. We identify non-canonical roles of RAL GTPases not as RAS effectors, but rather by acting upstream of RAS activation via induction of EGFR internalisation . Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to impacting stem cell proliferation and damage-induced intestinal regeneration, this function of RAL GTPases drives EGFR-dependent tumorigenic growth in the intestine and in human mammary epithelium. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of EGFR-driven tissue homeostasis and malignant transformation. Results: RalA is required within ISCs to induce midgut adult midgut regeneration following damage by oral infection with Erwinia carotovora carotovora 15 (Ecc15) (Johansson et al., 2019). To achieve a global view of intestinal pathways affected by RalA, we performed a transcriptomic analysis by RNAseq of whole midguts from vehicle treated (Mock) or damaged (Ecc15 fed) control animals or following RalA knockdown in intestinal stem and progenitor cells using the escargot-gal4 driver (ISC/EB>) (Micchelli and Perrimon, 2006). Consistent with its effect on ISC proliferation (Johansson et al., 2019), RalA knockdown significantly impaired damage-induced upregulation of cell cycle genes in the midgut. Additionally, levels of multiple transcriptional targets of the EGFR/MAPK pathway (Golembo et al., 1996; Hsu et al., 2001; Jin et al., 2015; Meng and Biteau, 2015), such as argos (aos), rhomboid (rho), Sox21a and string (stg) were increased following Ecc15 infection in control midguts. The upregulation of these target genes was significantly impaired upon RalA knockdown.
2021-02-01 | GSE162421 | GEO
Project description:we established a human intestinal stem cell (ISC) culture method expanded under feeder-free and fully defined conditions through selective enrichment of ISC populations (ISC3D-hIO) within hIO derived from human pluripotent stem cells.
2023-11-20 | GSE219017 | GEO
Project description:we established a human intestinal stem cell (ISC) culture method expanded under feeder-free and fully defined conditions through selective enrichment of ISC populations (ISC3D-hIO) within hIO derived from human pluripotent stem cells.
2023-11-20 | GSE219016 | GEO