Probes for LGR5
ACD can configure probes for the various manual and automated assays for LGR5 for RNAscope Assay, or for Basescope Assay compatible for your species of interest.
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EMBO j
2019 Dec 23
Novellasdemunt L, Kucharska A, Jamieson C, Prange-Barczynska M, Baulies A, Antas P, van der Vaart J, Gehart H, Maurice MM, Li VS
PMID: 31867777 | DOI: 10.15252/embj.2019102771
The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post-translational regulation of LGR5. Here, we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/?-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression.
Poultry Science
2023 Jan 01
Cloft, S;Uni, Z;Wong, E;
| DOI: 10.1016/j.psj.2023.102495
Mature small intestines have crypts populated by stem cells which produce replacement cells to maintain the absorptive villus surface area. The embryonic crypt is rudimentary and cells along the villi are capable of proliferation. By 7 d post-hatch the crypts are developed and are the primary sites of proliferation. Research characterizing the proliferative expansion of the small intestine during the peri-hatch period is lacking. The objective of this study was to profile the changes of genes that are markers of stem cells and proliferation: Olfactomedin 4 (Olfm4), Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and marker of proliferation Ki67 from embryonic day 17 to 7 d post-hatch using quantitative PCR and in situ hybridization (ISH). The expression of the stem cell marker genes differed. Olfm4 mRNA increased while Lgr5 mRNA decreased post-hatch. Ki67 mRNA decreased post-hatch in the duodenum and was generally the greatest in the ileum. The ISH was consistent with the quantitative PCR results. Olfm4 mRNA was only seen in the crypts and increased with morphological development of the crypts. In contrast Lgr5 mRNA was expressed in the crypt and the villi in the embryonic periods but became restricted to the intestinal crypt during the post-hatch period. Ki67 mRNA was expressed throughout the intestine pre-hatch, but then expression became restricted to the crypt and the center of the villi. The ontogeny of Olfm4, Lgr5 and Ki67 expressing cells show that proliferation in the peri-hatch intestine changes from along the entire villi to being restricted within the crypts.
EMBO Rep
2020 Feb 13
Pereira B, Amaral AL, Dias A, Mendes N, Muncan V, Silva AR, Thibert C, Radu AG, David L, M�ximo V, van den Brink GR, Billaud M, Almeida R
PMID: 32052574 | DOI: 10.15252/embr.201948938
Intestinal stem cells (ISCs) fuel the lifelong self-renewal of the intestinal tract and are paramount for epithelial repair. In this context, the Wnt pathway component LGR5 is the most consensual ISC marker to date. Still, the effort to better understand ISC identity and regulation remains a challenge. We have generated a Mex3a knockout mouse model and show that this RNA-binding protein is crucial for the maintenance of the Lgr5+ ISC pool, as its absence disrupts epithelial turnover during postnatal development and stereotypical organoid maturation ex vivo. Transcriptomic profiling of intestinal crypts reveals that Mex3a deletion induces the peroxisome proliferator-activated receptor (PPAR) pathway, along with a decrease in Wnt signalling and loss of the Lgr5+ stem cell signature. Furthermore, we identify PPAR? activity as a molecular intermediate of MEX3A-mediated regulation. We also show that high PPAR? signalling impairs Lgr5+ ISC function, thus uncovering a new layer of post-transcriptional regulation that critically contributes to intestinal homeostasis
Neurotensin regulates proliferation and stem cell function in the small intestine in a nutrient-dependent manner
Cellular and molecular gastroenterology and hepatology
2021 Sep 21
Rock, SA;Jiang, K;Wu, Y;Liu, Y;Li, J;Weiss, HL;Wang, C;Jia, J;Gao, T;Evers, BM;
PMID: 34560309 | DOI: 10.1016/j.jcmgh.2021.09.006
Intestinal stem cells (ISCs) are sensitive to dietary alterations and nutrient availability. Neurotensin (NT), a gut peptide localized predominantly to the small bowel and released by fat ingestion, stimulates the growth of intestinal mucosa under basal conditions and during periods of nutrient deprivation, suggesting a possible role for NT on ISC function.Lgr5-EGFP, NT wild type (Nt+/+) and Lgr5-EGFP, NT knockout (Nt-/-) mice were fed ad libitum (AL) or fasted for 48 h. Small intestine tissue and crypts were examined by gene expression analyses, fluorescence-activated cell sorting, western blot, immunohistochemistry, and crypt-derived organoid culture. Drosophila expressing NT in midgut enteroendocrine cells were fed a standard diet or low-energy diet and esg-GFP+ ISCs quantified via immunofluorescence.Loss of NT impaired crypt cell proliferation and ISC function in a manner dependent on nutrient status. Under nutrient-rich conditions, NT stimulated ERK1/2 signaling and the expression of genes that promote cell cycle progression, leading to crypt cell proliferation. Under conditions of nutrient depletion, NT stimulated WNT/ -catenin signaling and promoted an ISC gene signature, leading to enhanced ISC function. NT was required for the induction of WNT/ -catenin signaling and ISC-specific gene expression during nutrient depletion, and loss of NT reduced crypt cell proliferation and impaired ISC function and Lgr5 expression in the intestine during fasting. Conversely, the expression of NT in midgut enteroendocrine cells of Drosophila prevented loss of ISCs during nutrient depletion.Collectively, our findings establish an evolutionarily conserved role for NT in ISC maintenance during nutritional stress.
Cell reports
2022 Dec 13
Huang, XT;Li, T;Li, T;Xing, S;Tian, JZ;Ding, YF;Cai, SL;Yang, YS;Wood, C;Yang, JS;Yang, WJ;
PMID: 36516755 | DOI: 10.1016/j.celrep.2022.111796
Intestinal epithelial replenishment is fueled by continuously dividing intestinal stem cells (ISCs) resident at the crypt niche. However, the cell type(s) enabling replenishment upon damage and subsequent loss of whole crypts remain largely unclear. Using Set domain-containing protein 4 (Setd4), we identify a small population with reserve stem cell characteristics in the mouse intestine. Upon irradiation-induced injury, Setd4-expressing (Setd4+) cells survive radiation exposure and then activate to produce Sca-1-expressing cell types to restore the epithelial wall and regenerate crypts de novo via crypt fission. Setd4+ cells are confirmed to originate from the early fetal period, subsequently contributing to the development of embryonic gut and the establishment of postnatal crypts. Setd4+ cells are therefore represented as both originators and key regenerators of the intestine.
Cell Mol Gastroenterol Hepatol.
2020 Mar 05
Montenegro-Miranda PS, van der Meer JHM, Jones C, Meisner S, Vermeulen JLM, Koster J, Wildenberg ME, Heijmans J, Boudreau F, Ribeiro A, van den Brink GR, Muncan V
PMID: 32145468 | DOI: 10.1016/j.jcmgh.2020.02.007
BACKGROUND & AIMS:
Recent evidence has suggested that the intact intestinal epithelial barrier protects our body from a range of immune-mediated diseases. The epithelial layer has an impressive ability to reconstitute and repair upon damage and this process of repair increasingly is seen as a therapeutic target. In vitro models to study this process in primary intestinal cells are lacking.
METHODS:
We established and characterized an in vitro model of intestinal damage and repair by applying ?-radiation on small-intestinal organoids. We then used this model to identify novel regulators of intestinal regeneration.
RESULTS:
We identified hepatocyte nuclear factor 4? (HNF4?) as a pivotal upstream regulator of the intestinal regenerative response. Organoids lacking Hnf4a were not able to propagate in vitro. Importantly, intestinal Hnf4a knock-out mice showed impaired regeneration after whole-body irradiation, confirming intestinal organoids as a valuable alternative to in vivo studies.
CONCLUSIONS:
In conclusion, we established and validated an in vitro damage-repair model and identified HNF4? as a crucial regulator of intestinal regeneration
Stem cell reports
2022 Dec 29
Novellasdemunt, L;Kucharska, A;Baulies, A;Hutton, C;Vlachogiannis, G;Repana, D;Rowan, A;Suárez-Bonnet, A;Ciccarelli, F;Valeri, N;Li, VSW;
PMID: 36669491 | DOI: 10.1016/j.stemcr.2022.12.013
Adenomatous polyposis coli (APC) mutation is the hallmark of colorectal cancer (CRC), resulting in constitutive WNT activation. Despite decades of research, targeting WNT signaling in cancer remains challenging due to its on-target toxicity. We have previously shown that the deubiquitinating enzyme USP7 is a tumor-specific WNT activator in APC-truncated cells by deubiquitinating and stabilizing β-catenin, but its role in gut tumorigenesis is unknown. Here, we show in vivo that deletion of Usp7 in Apc-truncated mice inhibits crypt hyperproliferation and intestinal tumor development. Loss of Usp7 prolongs the survival of the sporadic intestinal tumor model. Genetic deletion, but not pharmacological inhibition, of Usp7 in Apc+/- intestine induces colitis and enteritis. USP7 inhibitor treatment suppresses growth of patient-derived cancer organoids carrying APC truncations in vitro and in xenografts. Our findings provide direct evidence that USP7 inhibition may offer a safe and efficacious tumor-specific therapy for both sporadic and germline APC-mutated CRC.
Research square
2023 Jan 10
Imada, S;Shin, H;Khawaled, S;Meckelmann, S;Whittaker, C;Correa, R;Pradhan, D;Calibasi, G;Nascentes, LN;Allies, G;Wittenhofer, P;Schmitz, O;Roper, J;Vinolo, M;Cheng, CW;Tasdogan, A;Yilmaz, ÃM;
PMID: 36711807 | DOI: 10.21203/rs.3.rs-2320717/v1
For more than a century, fasting regimens have improved health, lifespan, and tissue regeneration in diverse organisms, including humans. However, how fasting and post-fast refeeding impact adult stem cells and tumour formation has yet to be explored in depth. Here, we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation: Post-fast refeeding augments the regenerative capacity of Lgr5+ intestinal stem cells (ISCs), and loss of the tumour suppressor Apc in ISCs under post-fast refeeding leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum (AL) fed states. This demonstrates that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust induction of mTORC1 in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production, or protein synthesis abrogates the regenerative or tumourigenic effects of post-fast refeeding. Thus, fast-refeeding cycles must be carefully considered when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst not only in stem cell-driven regeneration but also in tumourigenicity.
The circadian clock gene, Bmal1, regulates intestinal stem cell signaling and represses tumor initiation
Cellular and molecular gastroenterology and hepatology
2021 Sep 14
Stokes, K;Nunes, M;Trombley, C;Flôres, DEFL;Wu, G;Taleb, Z;Alkhateeb, A;Banskota, S;Harris, C;Love, OP;Khan, WI;Rueda, L;Hogenesch, JB;Karpowicz, P;
PMID: 34534703 | DOI: 10.1016/j.jcmgh.2021.08.001
Circadian rhythms are daily physiological oscillations driven by the circadian clock: a 24-hour transcriptional timekeeper that regulates hormones, inflammation, and metabolism. Circadian rhythms are known to be important for health, but whether their loss contributes to colorectal cancer is not known.We tested the non-redundant clock gene, Bmal1, in intestinal homeostasis and tumorigenesis, using the Apcmin model of colorectal cancer.Bmal1 mutant, epithelium-conditional Bmal1 mutant, and photoperiod-disrupted mice bearing the Apcmin allele were assessed for tumorigenesis. Tumors and normal non-transformed tissue were characterized. Intestinal organoids were assessed for circadian transcription rhythms by RNA-sequencing, and in vivo and organoid assays were used to test Bmal1-dependent proliferation and self-renewal.Loss of Bmal1 or circadian photoperiod increases tumor initiation. In the intestinal epithelium the clock regulates transcripts involved in regeneration and intestinal stem cell signaling. Tumors have no self-autonomous clock function and only weak clock function in vivo. Apcmin clock-disrupted tumors exhibit high Yap (Hippo signaling) activity but exhibit low Wnt activity. Intestinal organoid assays reveal that loss of Bmal1 increases self-renewal in a Yap-dependent manner.Bmal1 regulates intestinal stem cell pathways, including Hippo signaling, and the loss of circadian rhythms potentiates tumor initiation.
Diet-induced alteration of intestinal stem cell function underlies obesity and prediabetes in mice
Nature metabolism
2021 Sep 01
Aliluev, A;Tritschler, S;Sterr, M;Oppenländer, L;Hinterdobler, J;Greisle, T;Irmler, M;Beckers, J;Sun, N;Walch, A;Stemmer, K;Kindt, A;Krumsiek, J;Tschöp, MH;Luecken, MD;Theis, FJ;Lickert, H;Böttcher, A;
PMID: 34552271 | DOI: 10.1038/s42255-021-00458-9
Excess nutrient uptake and altered hormone secretion in the gut contribute to a systemic energy imbalance, which causes obesity and an increased risk of type 2 diabetes and colorectal cancer. This functional maladaptation is thought to emerge at the level of the intestinal stem cells (ISCs). However, it is not clear how an obesogenic diet affects ISC identity and fate. Here we show that an obesogenic diet induces ISC and progenitor hyperproliferation, enhances ISC differentiation and cell turnover and changes the regional identities of ISCs and enterocytes in mice. Single-cell resolution of the enteroendocrine lineage reveals an increase in progenitors and peptidergic enteroendocrine cell types and a decrease in serotonergic enteroendocrine cell types. Mechanistically, we link increased fatty acid synthesis, Ppar signaling and the Insr-Igf1r-Akt pathway to mucosal changes. This study describes molecular mechanisms of diet-induced intestinal maladaptation that promote obesity and therefore underlie the pathogenesis of the metabolic syndrome and associated complications.
Translation initiation factor eIF2Bε promotes Wnt-mediated clonogenicity and global translation in intestinal epithelial cells
Stem cell research
2021 Aug 11
Smit, WL;de Boer, RJ;Meijer, BJ;Spaan, CN;van Roest, M;Koelink, PJ;Koster, J;Dekker, E;Abbink, TEM;van der Knaap, MS;van den Brink, GR;Muncan, V;Heijmans, J;
PMID: 34399164 | DOI: 10.1016/j.scr.2021.102499
Modulation of global mRNA translation, which is essential for intestinal stem cell function, is controlled by Wnt signaling. Loss of tumor supressor APC in stem cells drives adenoma formation through hyperactivion of Wnt signaling and dysregulated translational control. It is unclear whether factors that coordinate global translation in the intestinal epithelium are needed for APC-driven malignant transformation. Here we identified nucleotide exchange factor eIF2Bε as a translation initiation factor involved in Wnt-mediated intestinal epithelial stemness. Using eIF2BεArg191His mice with a homozygous point mutation that leads to dysfunction in the enzymatic activity, we demonstrate that eIF2Bε is involved in small intestinal crypt formation, stemness marker expression, and secreted Paneth cell-derived granule formation. Wnt hyperactivation in ex vivo eIF2BεArg191His organoids, using a GSK3β inhibitor to mimic Apc driven transformation, shows that eIF2Bε is essential for Wnt-mediated clonogenicity and associated increase of the global translational capacity. Finally, we observe high eIF2Bε expression in human colonic adenoma tissues, exposing eIF2Bε as a potential target of CRC stem cells with aberrant Wnt signaling.
An unsupervised method for physical cell interaction profiling of complex tissues
Nature methods
2021 Jul 12
Andrews, N;Serviss, JT;Geyer, N;Andersson, AB;Dzwonkowska, E;Šutevski, I;Heijboer, R;Baryawno, N;Gerling, M;Enge, M;
PMID: 34253926 | DOI: 10.1038/s41592-021-01196-2
Cellular identity in complex multicellular organisms is determined in part by the physical organization of cells. However, large-scale investigation of the cellular interactome remains technically challenging. Here we develop cell interaction by multiplet sequencing (CIM-seq), an unsupervised and high-throughput method to analyze direct physical cell-cell interactions between cell types present in a tissue. CIM-seq is based on RNA sequencing of incompletely dissociated cells, followed by computational deconvolution into constituent cell types. CIM-seq estimates parameters such as number of cells and cell types in each multiplet directly from sequencing data, making it compatible with high-throughput droplet-based methods. When applied to gut epithelium or whole dissociated lung and spleen, CIM-seq correctly identifies known interactions, including those between different cell lineages and immune cells. In the colon, CIM-seq identifies a previously unrecognized goblet cell subtype expressing the wound-healing marker Plet1, which is directly adjacent to colonic stem cells. Our results demonstrate that CIM-seq is broadly applicable to unsupervised profiling of cell-type interactions in different tissue types.
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| Description | ||
|---|---|---|
| sense Example: Hs-LAG3-sense | Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe. | |
| Intron# Example: Mm-Htt-intron2 | Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection | |
| Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G) | A mixture of multiple probe sets targeting multiple genes or transcripts | |
| No-XSp Example: Hs-PDGFB-No-XMm | Does not cross detect with the species (Sp) | |
| XSp Example: Rn-Pde9a-XMm | designed to cross detect with the species (Sp) | |
| O# Example: Mm-Islr-O1 | Alternative design targeting different regions of the same transcript or isoforms | |
| CDS Example: Hs-SLC31A-CDS | Probe targets the protein-coding sequence only | |
| EnEm | Probe targets exons n and m | |
| En-Em | Probe targets region from exon n to exon m | |
| Retired Nomenclature | ||
| tvn Example: Hs-LEPR-tv1 | Designed to target transcript variant n | |
| ORF Example: Hs-ACVRL1-ORF | Probe targets open reading frame | |
| UTR Example: Hs-HTT-UTR-C3 | Probe targets the untranslated region (non-protein-coding region) only | |
| 5UTR Example: Hs-GNRHR-5UTR | Probe targets the 5' untranslated region only | |
| 3UTR Example: Rn-Npy1r-3UTR | Probe targets the 3' untranslated region only | |
| Pan Example: Pool | A mixture of multiple probe sets targeting multiple genes or transcripts | |
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