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Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging.

ABSTRACT: The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt. In addition to the Lgr5 marker, intestinal stem cells have been associated with other markers that are expressed heterogeneously within the crypt base region. Previous quantitative clonal fate analyses have led to the proposal that homeostasis occurs as the consequence of neutral competition between dividing stem cells. However, the short-term behaviour of individual Lgr5(+) cells positioned at different locations within the crypt base compartment has not been resolved. Here we establish the short-term dynamics of intestinal stem cells using the novel approach of continuous intravital imaging of Lgr5- Confetti mice. We find that Lgr5(+) cells in the upper part of the niche (termed 'border cells') can be passively displaced into the transit-amplifying domain, after the division of proximate cells, implying that the determination of stem-cell fate can be uncoupled from division. Through quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed 'central cells', experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5(+) cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem-cell maintenance in which a dynamically heterogeneous cell population is able to function long term as a single stem-cell pool.

SUBMITTER: Ritsma L

PROVIDER: S-EPMC3964820 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging.

Ritsma Laila L Ellenbroek Saskia I J SIJ Zomer Anoek A Snippert Hugo J HJ de Sauvage Frederic J FJ Simons Benjamin D BD Clevers Hans H van Rheenen Jacco J

Nature 20140216 7492

The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt. In addition to the Lgr5 marker, intestinal stem cells have been associated with other markers that are expressed heterogeneously within the crypt base region. Previous quantitative clonal fate analyses have led to the proposal that homeostasis occurs as the consequence of neutral competition between dividing stem cells. However, the short-term behaviour of individual Lgr5(+) ...[more]

PMID: 24531760

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Project description:Background & aimsp21-activated kinase-1 (PAK1) belongs to a family of serine-threonine kinases and contributes to cellular pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and Wingless-related integration site(Wnt)/β-catenin, all of which are involved in intestinal homeostasis. Overexpression of PAK1 is linked to inflammatory bowel disease as well as colitis-associated cancer (CAC), and similarly was observed in interleukin (IL)10 knockout (KO) mice, a model of colitis and CAC. Here, we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL10 KO mice.MethodsIL10/PAK1 double-knockout (DKO) mice were generated and development of colitis and CAC was analyzed. Large intestines were measured and prepared for histology or RNA isolation. Swiss rolls were stained with H&E and periodic acid-Schiff. Co-immunoprecipitation and immunofluorescence were performed using intestinal organoids, SW480, and normal human colon epithelial cells 1CT.ResultsWhen compared with IL10 KO mice, DKOs showed longer colons and prolonged crypts, despite having higher inflammation and numbers of dysplasia. Crypt hyperproliferation was associated with Notch1 activation and diminished crypt differentiation, indicated by a reduction of goblet cells. Gene expression analysis indicated up-regulation of the Notch1 target hairy and enhancer of split-1 and the stem cell receptor leucin-rich repeat-containing G-protein-coupled receptor 5 in DKO mice. Interestingly, the stem cell marker olfactomedin-4 was present in colonic tissue. Increased β-catenin messenger RNA and cytoplasmic accumulation indicated aberrant Wnt signaling. Co-localization and direct interaction of Notch1 and PAK1 was found in colon epithelial cells. Notch1 activation abrogated this effect whereas silencing of PAK1 led to Notch1 activation.ConclusionsPAK1 contributes to the regulation of crypt homeostasis under inflammatory conditions by controlling Notch1. This identifies a novel PAK1-Notch1 axis in intestinal pathophysiology of inflammatory bowel disease and CAC.

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Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging.

Publications

Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging.

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