Wnt/planar cell polarity primed intestinal stem cells directly differentiate into enteroendocrine or Paneth cells [MoGene-2_0-st]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE94090 | GEO | 2020/09/11
REPOSITORIES: GEO
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Project description:A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required for better treatment options for a variety of chronic intestinal diseases, however, current models of ISC lineage hierarchy and segregation are still under debate. Here we report the identification of Lgr5+ ISCs that express Flattop (Fltp), a Wnt/planar cell polarity (PCP) reporter and effector gene. Functional analysis and lineage tracing revealed that Wnt/PCP-activated Fltp+ ISCs are primed towards either the enteroendocrine or Paneth cell lineage in vivo, while retaining self-renewal and multi-lineage capacity in vitro. Surprisingly, canonical Wnt/beta-catenin- and non-canonical Wnt/PCP-activated Lgr5+ ISCs are indistinguishable by the expression of stem-cell signature or secretory lineage-specifying genes, suggesting that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch of canonical to non-canonical Wnt signalling. Pseudotemporal ordering of targeted single-cell gene expression data allowed us to delineate the ISC differentiation path into enteroendocrine and Paneth cells. Strikingly, both lineages are directly recruited from ISCs via unipotent transition states, excluding the existence of formerly predicted bi- or multipotent secretory progenitors. Transitory cells that mature into Paneth cells are defined by label-retention and co-expression of stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ label-retaining cells (LRCs). Taken together, we identified the Wnt/PCP pathway as a new niche signal and polarity cue regulating stem cell fate. Active Wnt/PCP signalling represents one of the earliest events in ISC lineage priming towards the Paneth and enteroendocrine cell fate, preceding lateral inhibition and expression of secretory lineage-specifying genes. Thus, our findings provide a better understanding of the niche signals and redefine the mechanisms underlying ISC lineage hierarchy and segregation. Here we establish the Wnt/Planar cell polarity (PCP) gene Flattop (Fltp) as a unique marker for intestinal LRCs and their distinct secretory fate. We show that Fltp+ cells are characterized by a combined stem cell and secretory gene signature. A subset of Fltp+ cells is classified by label-retention and predominantly locates at position +4, indicative of quiescent stem cells. Strikingly, Fltp+ LRCs are specified by Wnt/PCP signaling in contrast to actively cycling stem cells that rely on the canonical Wnt/β-catenin pathway. In mice with disturbed Wnt/PCP signaling, the differentiation of enteroendocrine cells from LRCs is impaired. These findings establish Fltp as a novel marker for intestinal LRCs and implicate Wnt/PCP signaling in cell-cycle exit and commitment of ISCs to the secretory lineage. Taken together, we not only provide a marker to study LRCs in homeostatic and diseased conditions, but also identify the Wnt/PCP signaling pathway as a therapeutic target for colorectal cancer and metabolic disease.
2020-09-11 | GSE72112 | 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: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:The currently accepted intestinal epithelial cell organization model equates crypt base columnar (CBC) cells, marked by high levels of Lgr5 expression, with the intestinal stem cell (ISC). However, recent intestinal regeneration studies have uncovered limitations of the ‘Lgr5-CBC’ model, leading to two major views: one favoring the presence of a quiescent reserve stem cell population, the other calling for differentiated cell plasticity. To test if an alternative model may help reconcile these perspectives, we studied the hierarchical organization of crypt epithelial cells in an unbiased fashion, by combining high-resolution, single-cell profiling and lineage tracing in multiple transgenic mouse models. These show that Lgr5 is not a specific ISC marker; rather, cells located in the crypt isthmus, which include Lgr5low cells, comprise the ISCs that sustain tissue homeostasis. Following irradiation or intestinal injury, surviving ISCs and progenitors, but not differentiated cells, participate in intestinal regeneration, suggesting that neither de-differentiation nor reserve stem cell populations are drivers of intestinal regeneration. Our results provide a novel viewpoint for the intestinal crypt epithelium, in which ISCs localize to the crypt isthmus, and ISC potential is restricted to stem and progenitor cells.
2024-03-24 | GSE205229 | 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 intestinal epithelium is continuously regenerated by highly proliferative Lgr5+ intestinal stem cells (ISCs). The existence of a population of quiescent ISCs has been suggested yet its identity and features remain controversial. Here we describe that the expression of the RNA-binding protein Mex3a labels a subpopulation of Lgr5+ cells that divide less frequently and contribute to regenerate all intestinal lineages with slow kinetics. Single cell transcriptomic analysis revealed two classes of Lgr5-high cells, one of them defined by the Mex3a-expression program and by low levels of proliferation genes. Lineage tracing experiments show that large fraction of Mex3a+ cell population is continuously recalled into the rapidly dividing self-renewing ISC pool in homeostatic conditions. Chemotherapy and radiation target preferentially rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, which helps sustain the renewal of the intestinal epithelium during treatment.
2017-03-14 | GSE90856 | GEO
Project description:Using EphB2 or the ISC marker Lgr5, we have FACS-purified and profiled intestinal stem cells (ISCs), crypt proliferative progenitors and late transient amplifying cells to define a gene expression program specific for normal ISCs. A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. The intestinal stem cell signature predicts disease relapse in CRC and identifies a stem cell-like population that displays robust tumor- initiating capacity in immunodeficient mice as well as long-term self-renewal potential. We FACS purified mouse intestinal crypt cells according to their EphB2 or Lgr5 contents. We used Affymetrix chips to hybridize 2 samples from EphB2 high, 2 samples from EphB2 medium and 2 samples from EphB2 low cells (one sample from each group in a first hybridization on February 2009 plus an additional sample from each group on March 2009). Additionally, we hybridized one sample from Lgr5-EGFP high and one sample from Lgr5-EGFP low cells, obtained from Lgr5-EGFP knock-in mice (Barker et al., 2007).
2011-03-14 | E-GEOD-27605 | biostudies-arrayexpress
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