Gene expression in WT and Stat6 KO intestinal stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE123353 | GEO | 2018/12/05
REPOSITORIES: GEO
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Project description:The Lgr5 receptor is a marker of intestinal stem cells (ISCs) that regulates Wnt/b-catenin signaling. In this study, phenotype analysis of knockin/knockout Lgr5-eGFP-IRES-Cre and Lgr5-DTReGFP embryos revealed that Lgr5 deficiency during Wnt-mediated cytodifferentiation results in amplification of ISCs and early differentiation into Paneth cells, which can be counteracted by in utero treatment with the Wnt inhibitor LGK974. Conditional ablation of Lgr5 postnatally, but not in adults, altered stem cell fate towards the Paneth lineage. Together, these in vivo studies suggest that Lgr5 is part of a feedback loop to adjust the Wnt tone in ISCs. Moreover, transcriptome analyses revealed that Lgr5 controls fetal ISC maturation associated with acquisition of a definitive stable epithelial phenotype, that depends on the capacity of ISCs to generate their own extracellular matrix. Finally, using the ex vivo culture system, evidences are provided that Lgr5/Rspondin 2 interaction negatively regulates the pool of ISCs in organoids, revealing a sophisticated regulatory process for Wnt signaling in ISC.
2020-05-29 | GSE135362 | GEO
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:We demonstrate that Bmi1-expressing cells represent the predominant epithelial ISC in early intestinal development and precede existence of the canonical Wnt-dependent Lgr5+ stem cell population. We reveal that early in development, Bmi1+ ISCs are highly proliferative, then transition to a slow-cycling state with the emergence of the Lgr5+ ISC. Combining single cell RNA-sequencing (scRNA-seq) with bulk population ATAC-seq (Assay for Transposase Accessible Chromatin sequencing) analyses, we identify that a non-canonical Wnt signaling pathway correlates with the temporal presence of the highly proliferative developmental Bmi1+ ISC population.
2022-09-09 | GSE212798 | GEO
Project description:We used unbiased whole genome bisulfite sequencing (WGBS) to identify DNA methylation changes in the intestinal stem cells (ISCs) or their progeny during the suckling period of mouse colon development. Lgr5-EGFP mice were used to identify ISC populations in the colons. WGBS were performed using EGFP labeled Lgr5+ ISCs and epithelial cell adhesion molecule (EpCAM) labeled epithelial cells isolated at the beginning and end of the suckling period (postnatal day 0-P0 and P21).
2015-08-25 | E-GEOD-58532 | biostudies-arrayexpress
Project description:We used RNA sequencing to quantify the gene expression levels in the intestinal stem cells (ISCs) or their progeny during the suckling period of mouse colon development. Lgr5-EGFP mice were used to identify ISC populations in the colons. RNA sequencing was performed using EGFP labeled Lgr5+ ISCs and epithelial cell adhesion molecule (EpCAM) labeled epithelial cells isolated at the beginning and end of the suckling period (postnatal day 0-P0 and P21).
2015-08-25 | E-GEOD-64054 | biostudies-arrayexpress
Project description:Purpose: circRNAs emerge as critical modulators in various biogical processes, but their roles in intestinal stem cells (ISC) reamin unclear. Here we want to identify a functional circRNA in ISCs, and firstly, we explore the expression profile of circRNA in ISCs. Methods: We isolated Lgr5+ ISCs and Lgr5- non-ISCs from the intestine tissue, followed by circRNA seq. Results: Many circRNAs are differently expressed in ISCs and non-ISCs, and we selected circRNAs highly expressed in ISCs for further investigation. Conclusions: circRNAs may play a critical role in ISC self-renewal.
2018-11-29 | GSE123051 | GEO
Project description:The small intestine is a rapidly proliferating organ that is maintained by a small population of Lgr5-expressing intestinal stem cells (ISCs). However, several Lgr5-negative ISC populations have been identified, and this remarkable plasticity allows the intestine to rapidly respond to both the local environment and to damage. The mediators of such plasticity are still largely unknown. Using intestinal organoids and mouse models, we show that upon ribosome impairment (driven by Rptor deletion, amino acid starvation, or low dose cyclohexamide treatment) ISCs gain an Lgr5-negative, fetal-like identity. This is accompanied by a rewiring of metabolism. Our findings suggest that the ribosome can act as a sensor of nutrient availability, allowing ISCs to respond to the local nutrient environment. Mechanistically, we show that this phenotype requires the activation of ZAKɑ, which in turn activates YAP, via SRC. Together, our data reveals a central role for ribosome dynamics in intestinal stem cells, and identify the activation of ZAKɑ as a critical mediator of stem cell identity.
2022-06-13 | PXD033122 | Pride
Project description:Imbalance in intestinal stem cell (ISC) homeostasis leads to cancer and metabolic disease. Therefore intense efforts are underway to understand ISC hierarchy and the niche signals that control stem cell fate. Several ISC populations have been described according to their marker expression, cell-cycle behavior and lineage potential. Actively cycling Lgr5+ ISCs ensure homeostatic renewal. A less well-defined and minor population of quiescent and label-retaining cells (LRCs) is viewed as a reserve stem cell pool. However, the existence of quiescent stem cells distinct from the Lgr5+ ISCs is controversial. Indeed, recent findings identified quiescent intestinal cells as a subpopulation of Lgr5+ ISCs that are committed to the secretory fate, but the signals that maintain and activate quiescent cells/LRCs remain elusive.
2020-09-11 | GSE94090 | 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 stem cells (ISCs) depend on niche factors for proper function. Here, we focus on RSPO3, an essential ISC niche factor, that engages the Lgr5 receptor on ISCs to potentiate WNT signaling, an interaction that is critical for maintaining intestinal stemness. Leveraging novel Rspo3-GFP and Grem1-tdTomato-CreERT2 genetically engineered mice together with single-cell mRNA profiling, we find that RSPO3 is expressed by lymphatic endothelial cells (LECs) and Rspo3+Grem1+ (RG) fibroblasts in the intestinal stroma where RG fibroblasts surround lymphatics and are in close proximity to Lgr5+ ISCs near the crypt base. Functionally, RG fibroblasts through the production of RSPO3 and GREM1 and LECs through the production of RSPO3 foster intestinal organoid propagation in vitro. Rspo3 loss in either or both of these niche cells in vivo compromises ISC numbers, villi length, and repair after irradiation-induced injury. Mechanistically, irradiation-induced damage expands LEC and RG fibroblast numbers and enhances the latters’ generation of RSPO3 through IL-1 receptor activation. We propose that LECs represent a novel component of the ISC niche, which together with RG fibroblasts, provide essential RSPO3 to sustain ISCs in homeostasis and regeneration.
2022-09-08 | GSE185354 | GEO