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Transcriptomic profiling of small intestinal crypts upon MEX3A deletion

ABSTRACT: The effort to better understand intestinal stem cell (ISC) identity and regulation remains a challenge. We have been studying the RNA-binding protein MEX3A as a putative ISC marker. In that context, we have generated the first Mex3a knockout (KO) mouse model and show MEX3A is crucial for maintenance of the Lgr5+ ISC pool. As part of a phenotypic characterization pipeline, we have performed transcriptomic profiling (RNA-sequencing) of isolated Mex3a KO small intestinal crypts and compared it against small intestinal crypts isolated from age-matched wild-type controls.

ORGANISM(S): Mus musculus

PROVIDER: GSE141191 | GEO | 2020/02/13

REPOSITORIES: GEO

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Mex3a Marks a Slowly Dividing Subpopulation of Lgr5+ Intestinal Stem Cells

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.

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Transcriptomic profiling of small intestinal crypts upon MEX3A deletion

Project description:Transcriptomic profiling of small intestinal crypts upon MEX3A deletion

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Transcriptomic profilling of Mex3a+ cells in colon adenomas compared to LGR5

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Retrograde movements determine effective stem cell numbers in intestinal crypts

Project description:Our goal was to compare the transcriptomic profile of lgr5+ high, medium and low cells (corresponding to centre, border and >3 row in crypts) between small and large intestine in mice. We observed differences in the transcriptional profile between centre and border Lgr5+ cells within small and large intestinal crypts and more profound differences between the two intestinal sites.

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RNA-seq of crypt cells from mouse small intestinal epithelium with an intestinal-epithelial specific deletion of LSD1 compared to wild type crypts

Project description:To assess the role of LSD1 in mouse small intestinal epithelium, we isolated small intestinal crypts from wild type (WT) (Villin-Cre -; Lsd1f/f) and intestinal-epithelial-specific knock-out (KO) (Villin-Cre+; Lsd1f/f) mice. We dissociated crypts into single cells, and FACS sorted Epcam+ cells, to avoid immune-cell contamination. RNA was directly isolated from these sorted cells, and this was used for RNA seq. As KO crypts are different from WT crypts (KO crypts lack Paneth cells), identifying genes specifically regulated by LSD1 helps us to identify how LSD1 regulates intestinal crypt biology. Specifically, because we were able to combine this with ChIP-seq of the same cells, to identify where H3K4me1 levels (target of the histone demethylase LSD1) were different in the genome.

2020-11-16 | E-MTAB-7862 | 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

RNA-binding Mex3a promotes epithelial stemness and represses Klf4-dependent differentiation upon intestinal homeostasis, regeneration and cancer

Project description:The precise control of the self-renewal and differentiation of intestinal stem cells (ISCs) is essential for intestinal homeostasis, efficient regeneration and prevention of tumorigenesis. However, the underlying molecular mechanisms remain only partially understood, especially in regeneration and cancer. Here, it is demonstrated that RNA-binding protein Mex3a is specifically expressed in ISCs and promotes their stemness and proliferative status. Its depletion results in compromised ISCs, impaired intestinal regeneration and suppressed oncogenic transformation. Mechanistically, upregulation of Mex3a in ISCs at homeostasis, in postirradiation regenerative foci and in colorectal cancer model is regulated by the upstream transcription factor E2f3. In turn, Mex3a activates Wnt signaling by directly suppressing pro-differentiation transcription factor Klf4 at the mRNA level. Furthermore, expression of E2F3 and MEX3A significantly increases in human radiation enteritis and colorectal carcinoma (CRC), accompanied by KLF4 downregulation and Wnt signaling hyperactivation. These findings indicate that the E2f3-Mex3a-Klf4-Wnt as the critical molecular switch between ISC self-renewal and differentiation. It can represent a putative therapeutic target for CRC and regeneration-related gut diseases.

2021-07-09 | GSE179493 | GEO

Isolation of Mex3a / Lgr5 cell population in mouse small intestine

Project description:Proximal mouse small intestine from mice bearing the Lgr5 GFP/+ and Mex3a Tom/+ alleles were used to obtain single cell preparations. Cells were selected for GFP expression and different levels of tdTomato were defined. Sorted cells were lysed and processed for transcriptomic analysis

2017-06-01 | GSE90629 | GEO

scRNAseq of AKP Mex3a KO mouse colorectal organoids in FOLFIRI treatment

Project description:Mex3a is an RNA binding protein of unknown function. To elucidate the contribution of Mex3a to tumoral heterogeneity, Mex3a KO organoids engineered by CRISPR were sequenced in three different conditions. Live organoids (DAPI negative) were sorted in Control, after 2 days of FOLFIRI and after 5 days of treatment. Two WT organoids (parental and a derived clone) and two KO (KO1 and KO2, two independent clones) were used for this experiment.

2022-05-16 | E-MTAB-11146 | biostudies-arrayexpress

Gene expression data from crypts isolated from WT and Lpcat3 Villin-CreERT2 mice

Project description:We used microarray to analyze the global gene expression in crypts isolated from wild-type (Lpcat3F/F) and Lpcat3 tamoxifen inducible intestine-specific knockout (Lpcat3F/F, Villin-CreERT2) crypts. Phospholipid remodeling is a critical determinant of membrane composition and function. How membrane phospholipid composition affects tissue stem cell function and tumorigenesis is unknown. Here we demonstrate that Lpcat3-dependent phospholipid remodeling regulates intestinal stem cell (ISC) proliferation and promotes tumorigenesis in intestine. The objective of generating this dataset was to analyze the effects of Lpcat3 loss of function on gene expression in mouse intestine crypts.

2017-12-05 | GSE98516 | GEO

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