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ABSTRACT: The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation.
Nature 03/2013; 495(7439):65-9. · 36.28 Impact Factor
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Catherine H Wilson,
Catriona Crombie,
Louise van der Weyden,
George Poulogiannis,
Alistair G Rust,
Mercedes Pardo,
Tannia Gracia,
Lu Yu,
Jyoti Choudhary,
Gino B Poulin,
Rebecca E McIntyre, Douglas J Winton,
H Nikki March,
Mark J Arends,
Andrew G Fraser,
David J Adams
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ABSTRACT: Genetic screens in simple model organisms have identified many of the key components of the conserved signal transduction pathways that are oncogenic when misregulated. Here, we identify H37N21.1 as a gene that regulates vulval induction in let-60(n1046gf), a strain with a gain-of-function mutation in the Caenorhabditis elegans Ras orthologue, and show that somatic deletion of Nrbp1, the mouse orthologue of this gene, results in an intestinal progenitor cell phenotype that leads to profound changes in the proliferation and differentiation of all intestinal cell lineages. We show that Nrbp1 interacts with key components of the ubiquitination machinery and that loss of Nrbp1 in the intestine results in the accumulation of Sall4, a key mediator of stem cell fate, and of Tsc22d2. We also reveal that somatic loss of Nrbp1 results in tumourigenesis, with haematological and intestinal tumours predominating, and that nuclear receptor binding protein 1 (NRBP1) is downregulated in a range of human tumours, where low expression correlates with a poor prognosis. Thus NRBP1 is a conserved regulator of cell fate, that plays an important role in tumour suppression.
The EMBO Journal 04/2012; 31(11):2486-97. · 9.20 Impact Factor
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Catherine H Wilson,
Catriona Crombie,
Louise van der Weyden,
George Poulogiannis,
Alistair G Rust,
Mercedes Pardo,
Tannia Gracia,
Lu Yu,
Jyoti Choudhary,
Gino B Poulin,
Rebecca E McIntyre, Douglas J Winton,
H Nikki March,
Mark J Arends,
Andrew G Fraser,
David J Adams
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ABSTRACT: Genetic screens in simple model organisms have identified many of the key components of the conserved signal transduction pathways that are oncogenic when misregulated. Here, we identify
The EMBO Journal 04/2012; 31(11):2486-2497. · 9.20 Impact Factor
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H Nikki March,
Alistair G Rust,
Nicholas A Wright,
Jelle ten Hoeve,
Jeroen de Ridder,
Matthew Eldridge,
Louise van der Weyden,
Anton Berns,
Jules Gadiot,
Anthony Uren,
Richard Kemp,
Mark J Arends,
Lodewyk F A Wessels, Douglas J Winton,
David J Adams
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ABSTRACT: The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development.
Nature Genetics 11/2011; 43(12):1202-9. · 35.53 Impact Factor
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Gastroenterology 03/2011; 140(5):1387-91. · 11.68 Impact Factor
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ABSTRACT: The stem cells of the small intestine are multipotent: they give rise, via transit-amplifying cell divisions, to large numbers of columnar absorptive cells mixed with much smaller numbers of three different classes of secretory cells--mucus-secreting goblet cells, hormone-secreting enteroendocrine cells, and bactericide-secreting Paneth cells. Notch signaling is known to control commitment to a secretory fate, but why are the secretory cells such a small fraction of the population, and how does the diversity of secretory cell types arise? Using the mouse as our model organism, we find that secretory cells, and only secretory cells, pass through a phase of strong expression of the Notch ligand Delta1 (Dll1). Onset of this Dll1 expression coincides with a block to further cell division and is followed in much less than a cell cycle time by expression of Neurog3--a marker of enteroendocrine fate--or Gfi1--a marker of goblet or Paneth cell fate. By conditional knock-out of Dll1, we confirm that Delta-Notch signaling controls secretory commitment through lateral inhibition. We infer that cells stop dividing as they become committed to a secretory fate, while their neighbors continue dividing, explaining the final excess of absorptive over secretory cells. Our data rule out schemes in which cells first become committed to be secretory, and then diversify through subsequent cell divisions. A simple mathematical model shows how, instead, Notch signaling may simultaneously govern the commitment to be secretory and the choice between alternative modes of secretory differentiation.
PLoS ONE 01/2011; 6(9):e24484. · 4.09 Impact Factor
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ABSTRACT: With the capacity for rapid self-renewal and regeneration, the intestinal epithelium is stereotypical of stem cell-supported tissues. Yet the pattern of stem cell turnover remains in question. Applying analytical methods from population dynamics and statistical physics to an inducible genetic labeling system, we showed that clone size distributions conform to a distinctive scaling behavior at short times. This result demonstrates that intestinal stem cells form an equipotent population in which the loss of a stem cell is compensated by the multiplication of a neighbor, leading to neutral drift dynamics in which clones expand and contract at random until they either take over the crypt or they are lost. Combined with long-term clonal fate data, we show that the rate of stem cell replacement is comparable to the cell division rate, implying that neutral drift and symmetrical cell divisions are central to stem cell homeostasis.
Science 09/2010; 330(6005):822-5. · 31.20 Impact Factor
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Gabrielle H Ashton,
Jennifer P Morton,
Kevin Myant,
Toby J Phesse,
Rachel A Ridgway,
Victoria Marsh,
Julie A Wilkins,
Dimitris Athineos,
Vanesa Muncan,
Richard Kemp,
Kristi Neufeld,
Hans Clevers,
Valerie Brunton, Douglas J Winton,
Xiaoyan Wang,
Rosalie C Sears,
Alan R Clarke,
Margaret C Frame,
Owen J Sansom
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ABSTRACT: The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein within the intestinal epithelium. Following Apc loss, FAK expression increased in a c-Myc-dependent manner. Codeletion of Apc and Fak strongly reduced proliferation normally induced following Apc loss, and this was associated with reduced levels of phospho-Akt and suppression of intestinal tumorigenesis in Apc heterozygous mice. Thus, FAK is required downstream of Wnt Signaling, for Akt/mTOR activation, intestinal regeneration, and tumorigenesis. Importantly, this work suggests that FAK inhibitors may suppress tumorigenesis in patients at high risk of developing colorectal cancer.
Developmental cell 08/2010; 19(2):259-69. · 13.36 Impact Factor
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ABSTRACT: Reporter genes are widely used in biology and only a limited number are available. We present a new reporter gene for the localization of mammalian cells and transgenic tissues based on detection of the bglA (SYNbglA) gene of Caldocellum saccharolyticum that encodes a thermophilic beta-glucosidase.
SYNbglA was generated by introducing codon substitutions to remove CpG motifs as these are associated with gene silencing in mammalian cells. SYNbglA expression can be localized in situ or detected quantitatively in colorimetric assays and can be co-localized with E. coli beta-galactosidase. Further, we have generated a Cre-reporter mouse in which SYNbglA is expressed following recombination to demonstrate the general utility of SYNbglA for in vivo analyses. SYNbglA can be detected in tissue wholemounts and in frozen and wax embedded sections.
SYNbglA will have general applicability to developmental and molecular studies in vitro and in vivo.
BMC Biology 01/2010; 8:89. · 5.75 Impact Factor
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Jean-Leon Chong,
Pamela L Wenzel,
M Teresa Sáenz-Robles,
Vivek Nair,
Antoney Ferrey,
John P Hagan,
Yorman M Gomez,
Nidhi Sharma,
Hui-Zi Chen,
Madhu Ouseph, [......],
Brian Culp,
Louise Mezache, Douglas J Winton,
Owen J Sansom,
Danian Chen,
Rod Bremner,
Paul G Cantalupo,
Michael L Robinson,
James M Pipas,
Gustavo Leone
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ABSTRACT: In the established model of mammalian cell cycle control, the retinoblastoma protein (Rb) functions to restrict cells from entering S phase by binding and sequestering E2f activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examined the effects of E2f1, E2f2 and E2f3 triple deficiency in murine embryonic stem cells, embryos and small intestines. We show that in normal dividing progenitor cells E2f1-3 function as transcriptional activators, but contrary to the current view, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells E2f1-3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2f1-3 from repressors to activators, leading to the superactivation of E2f responsive targets and ectopic cell divisions. Loss of E2f1-3 completely suppressed these phenotypes caused by Rb deficiency. This work contextualizes the activator versus repressor functions of E2f1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles.
Nature 12/2009; 462(7275):930-4. · 36.28 Impact Factor
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ABSTRACT: Summary K-ras mutations are found in 40-50% of human colorectal adenomas and carcinomas, but their functional contribution remains incompletely understood. Here, we show that a conditional mutant K-ras mouse model (K-ras(Asp12)/Cre), with transient intestinal Cre activation by beta-Naphthoflavone (beta-NF) treatment, displayed transgene recombination and K-ras(Asp12) expression in the murine intestines, but developed few intestinal adenomas over 2 years. However, when crossed with Apc(Min/+) mice, the K-ras(Asp12)/Cre/Apc(Min/+) offspring showed acceleration of intestinal tumourigenesis with significantly changed average lifespan (P < 0.05) decreased to 18.4 +/- 5.4 weeks from 20.9 +/- 4.7 weeks (control Apc(Min/+) mice). The numbers of adenomas in the small intestine and large intestine were significantly (P < 0.01) increased by 1.5-fold and 5.7-fold, respectively, in K-ras(Asp12)/Cre/Apc(Min/+) mice compared with Apc(Min/+) mice, with the more marked increase in adenoma prevalence in the large intestine. To explore possible mechanisms for K-ras(Asp12) and Apc(Min) co-operation, the Mitogen-activated protein kinase (Mapk), Akt and Wnt signalling pathways, including selected target gene expression levels, were evaluated in normal large intestine and large intestinal tumours. K-ras(Asp12) increased activation of Mapk and Akt signalling pathway targets phospho-extracellular signal-regulated kinase (pErk) and pAkt, and increased relative expression levels of Wnt pathway targets vascular endothelial growth factor (VEGF), gastrin, cyclo-oxygenase 2 (Cox2) and T-cell lymphoma invasion and metastasis 1 (Tiam1) in K-ras(Asp12)/Cre/Apc(Min/+) adenomas compared with that of Apc(Min/+) adenomas, although other Wnt signalling pathway target genes such as Peroxisome proliferator-activated receptor delta (PPARd), matrix metalloproteinase 7 (MMP7), protein phosphatase 1 alpha (PP1A) and c-myc remained unchanged. In conclusion, intestinal expression of K-ras(Asp12) promotes mutant Apc-initiated intestinal adenoma formation in vivo more in the large intestine than the small intestine, with evidence of synergistic co-operation between mutant K-ras and Apc involving increased expression of some Wnt-pathway target genes.
International Journal of Experimental Pathology 10/2009; 90(5):558-74. · 2.57 Impact Factor
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ABSTRACT: The Lkb1 tumour suppressor is a multitasking kinase participating in a range of physiological processes. We have determined the impact of Lkb1 deficiency on intestinal homeostasis, particularly focussing on secretory cell differentiation and development since we observe strong expression of Lkb1 in normal small intestine Paneth and goblet cells. We crossed mice bearing an Lkb1 allele flanked with LoxP sites with those carrying a Cyp1a1-specific inducible Cre recombinase. Lkb1 was efficiently deleted from the epithelial cells of the mouse intestine after intraperitoneal injection of the inducing agent beta-naphthoflavone. Bi-allelic loss of Lkb1 led to the perturbed development of Paneth and goblet cell lineages. These changes were characterised by the lack of Delta ligand expression in Lkb1-deficient secretory cells and a significant increase in the levels of the downstream Notch signalling effector Hes5 but not Hes1. Our data show that Lkb1 is required for the normal differentiation of secretory cell lineages within the intestine, and that Lkb1 deficiency modulates Notch signalling modulation in post-mitotic cells.
PLoS ONE 02/2009; 4(1):e4264. · 4.09 Impact Factor
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ABSTRACT: Herpes simplex virus type 1 (HSV-1) has the capacity to establish a life-long latent infection in sensory neurones and also to periodically reactivate from these cells. Since mutant viruses defective for immediate-early (IE) expression retain the capacity for latency establishment it is widely assumed that latency is the consequence of a block in IE gene expression. However, it is not clear whether viral gene expression can precede latency establishment following wild-type virus infection. In order to address this question we have utilized a reporter mouse model system to facilitate a historical analysis of viral promoter activation in vivo. This system utilizes recombinant viruses expressing Cre recombinase under the control of different viral promoters and the Cre reporter mouse strain ROSA26R. In this model, viral promoter-driven Cre recombinase mediates a permanent genetic change, resulting in reporter gene activation and permanent marking of latently infected cells. The analyses of HSV-1 recombinants containing human cytomegalovirus major immediate-early, ICP0, gC or latency-associated transcript promoters linked to Cre recombinase in this system have revealed the existence of a population of neurones that have experienced IE promoter activation prior to the establishment of latency.
Journal of General Virology 01/2009; 89(Pt 12):2965-74. · 3.36 Impact Factor
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ABSTRACT: PTEN acts as a tumor suppressor in a range of tissue types and has been implicated in the regulation of intestinal stem cells. To study Pten function in the intestine, we used various conditional transgenic strategies to specifically delete Pten from the mouse intestinal epithelium. We show that Pten loss specifically within the adult or embryonic epithelial cell population does not affect the normal architecture or homeostasis of the epithelium. However, loss of Pten in the context of Apc deficiency accelerates tumorigenesis through increased activation of Akt, leading to rapid development of adenocarcinoma. We conclude that Pten is redundant in otherwise normal intestinal epithelium and epithelial stem cells but, in the context of activated Wnt signaling, suppresses progression to adenocarcinoma through modulation of activated Akt levels.
Nature Genetics 01/2009; 40(12):1436-44. · 35.53 Impact Factor
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ABSTRACT: According to the current model of adult epidermal homeostasis, skin tissue is maintained by two discrete populations of progenitor cells: self-renewing stem cells; and their progeny, known as transit amplifying cells, which differentiate after several rounds of cell division. By making use of inducible genetic labelling, we have tracked the fate of a representative sample of progenitor cells in mouse tail epidermis at single-cell resolution in vivo at time intervals up to one year. Here we show that clone-size distributions are consistent with a new model of homeostasis involving only one type of progenitor cell. These cells are found to undergo both symmetric and asymmetric division at rates that ensure epidermal homeostasis. The results raise important questions about the potential role of stem cells on tissue maintenance in vivo.
Nature 04/2007; 446(7132):185-9. · 36.28 Impact Factor
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ABSTRACT: Deregulation of Wnt signalling has recently been implicated in human renal cancer. Here, we directly test this association by using a Cre-LoxP strategy to inactivate the Adenomatous Polyposis Coli (Apc) gene in the murine renal epithelium. Mice homozygous for a conditional Apc allele were intercrossed with mice transgenic for Cre recombinase under control of the Cyp1A promoter, which delivers constitutive recombination within a proportion of cells in the renal epithelium. Inactivation of Apc leads to the accumulation of nuclear beta-catenin and the rapid development of multiple dysplastic foci. Renal carcinoma was observed with an earliest onset of 4 months. This predisposition was accelerated by p53 deficiency, reducing the earliest onset to 2 months. Compared to other murine models of kidney neoplasia, this represents particularly rapid onset of disease, and so implicates an important role for Apc in suppressing renal carcinoma.
Oncogene 01/2006; 24(55):8205-10. · 6.37 Impact Factor
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ABSTRACT: Here, we exploit an absolute differential between stem and progeny cells in their ability to express Cre from a somatically inducible transgene to determine the longevity of intestinal Paneth cells. In the Ahcre transgenic line induction of Cre recombinase allows constitutive activation of a Cre-activated reporter in intestinal precursors but not in Paneth cells. The time taken for Paneth cells to inherit the reporter (EYFP) was measured in adult Ahcre/R26R-EYFP animals. Using confocal microscopy of TOPRO-3-stained sections, both precursors and Paneth cells were identified and subsequently scored for EYFP expression. It takes up to 57 days for Paneth cells to inherit the reporter, making them three times longer-lived than previously indicated using nucleotide incorporation and suggesting that such determinations of cell turnover may be significant underestimates.
Developmental Dynamics 09/2005; 233(4):1332-6. · 2.54 Impact Factor
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ABSTRACT: Cyclin D1 is postulated to be a target of the canonical Wnt pathway and critical for intestinal adenoma development. We show here that, unlike cyclin D1 reporter assays, endogenous cyclin D1 levels are not affected following antagonism of the Wnt pathway in vitro, nor is cyclin D1 immediately up-regulated following conditional loss of Apc in vivo. Cyclin D1 levels do, however, increase in a delayed manner in a small subset of cells, suggesting such up-regulation occurs as a secondary event. We also analyzed the immediate consequences of Apc loss in a cyclin D1(-/-) background and failed to find any cyclin D1-dependent phenotypes. However, we did observe elevated cyclin D1 expression in lesions developing 20 days after Apc loss. In these circumstances, all adenomas (but not smaller lesions) showed cyclin D1 up-regulation. Finally in a smaller study, we analyzed whether cyclin D1 deficiency affected adenoma formation 20 days following induced loss of Apc. Unlike AhCre(+) Apc(fl/fl) mice (which all developed adenomas), doubly mutant AhCre(+) Apc(fl/fl) cyclin D1(-/-) mice only developed small lesions. Taken together, this argues that cyclin D1 up-regulation in intestinal neoplasia is important for tumor progression rather than initiation.
Journal of Biological Chemistry 09/2005; 280(31):28463-7. · 4.77 Impact Factor
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ABSTRACT: Based on recent reports that peroxisome proliferator-activated receptor delta (PPARdelta) activation promotes tumourigenesis, we have investigated the role of this protein in Apc-mediated intestinal tumourigenesis. We demonstrate that the inactivation of Apc in the adult small intestine, while causing the expected nuclear accumulation of beta-catenin, does not cause the expected increase in PPARdelta mRNA or protein but conversely, the levels of PPARdelta mRNA and protein are lowered. Furthermore, we find that ApcMinPPARdelta-null mice exhibit an increased predisposition to intestinal tumourigenesis. Our data suggest that PPARdelta is not directly regulated by beta-catenin, and that inhibition of PPARdelta activity is unlikely to be an appropriate strategy for the chemoprevention or chemotherapy of intestinal malignancies.
Oncogene 12/2004; 23(55):8992-6. · 6.37 Impact Factor
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ABSTRACT: Based on recent reports that peroxisome proliferator-activated receptor delta (PPAR) activation promotes tumourigenesis, we have investigated the role of this protein in Apc-mediated intestinal tumourigenesis. We demonstrate that the inactivation of Apc in the adult small intestine, while causing the expected nuclear accumulation of -catenin, does not cause the expected increase in PPAR mRNA or protein but conversely, the levels of PPAR mRNA and protein are lowered. Furthermore, we find that ApcMinPPAR-null mice exhibit an increased predisposition to intestinal tumourigenesis. Our data suggest that PPAR is not directly regulated by -catenin, and that inhibition of PPAR activity is unlikely to be an appropriate strategy for the chemoprevention or chemotherapy of intestinal malignancies.Keywords: PPARdelta, tumourigenesis, Apc, intestine, PPARbeta
Oncogene 10/2004; 23(55):8992-8996. · 6.37 Impact Factor
