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Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional <em>Cre-lox</em> Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Cardiff University

Expanding the p53 toolkit: a new platform for rapid engineering of the p53 locus in vivo.

Expression of the Wnt target gene ASCL2 is elevated in 78% of intestinal neoplasia datasets (Oncomine), suggesting a role for deregulated ASCL2 in the aetiology of intestinal tumourigenesis. Furthermore, ectopic expression of Ascl2 has previously been shown to lead to hyperplasia in the mouse. However, elevated levels of ASCL2 does not have an impact on the overall survival or recurrence-free survival rates in colorectal cancer patients. Here the authors use a novel mouse model to analyse the role of Ascl2 in intestinal tumourigenesis and address the controversy surrounding the relevance of this gene to the aetiology of colorectal cancer.

Entopic overexpression of Ascl2 does not accelerate tumourigenesis in ApcMin mice.

The identification of the intestinal stem cell (ISC) markers Lgr5 and Bmi-1 has furthered our understanding of how they accomplish homeostasis in this rapidly self-renewing tissue. Recent work indicates that these markers identify a cycling Lgr5(+) ISC which can be replaced by a quiescent Bmi-1(+) ISC. Currently, there is little data on how these cells interact to control intestinal crypt homeostasis and regeneration. This interaction likely involves other differentiated cells within the niche as it has previously been demonstrated that the "stemness" of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. To investigate this, we used two conditional mouse models to delete the transcription factor β-catenin within the intestinal crypt. Critically these differ in their ability to drive recombination within Paneth cells and therefore allow us to compare the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. After gene deletion, the intestines in the model in which Paneth cells were retained showed a rapid recovery and repopulation of the crypt-villus axis presumably from either a spared ISC or the hypothetical quiescent ISCs. However, in the absence of Paneth cells the recovery ability was compromised resulting in complete loss of intestinal epithelial integrity. This data indicates that the Paneth cells play a crucial role within the in vivo ISC niche in aiding recovery following substantial insult.

Evidence for a crucial role of paneth cells in mediating the intestinal response to injury.

Colorectal cancer (CRC) is the third most common cancer in the UK, with over 37,500 people being diagnosed every year. Survival rates for CRC have doubled in the last 30 years and it is now curable if diagnosed early, but still over half of all sufferers do not survive for longer than 5 years after diagnosis. The major complication to treating this disease is that of metastasis, specifically to the liver, which is associated with a 5 year survival of less than 5%. These statistics highlight the importance of the development of earlier detection techniques and more targeted therapeutics. The future of treating this disease therefore lies in increasing understanding of the mutations which cause tumourigenesis, and insight into the development and progression of this complex disease. This can only be achieved through the use of functional models which recapitulate all aspects of the human disease. There is a wide range of models of CRC available to researchers, but all have their own strengths and weaknesses. Here we review how CRC can be modelled and discuss the future of modelling this complex disease, with a particular focus on how genetically engineered mouse models have revolutionised this area of research.

What are the best routes to effectively model human colorectal cancer?

Brg1 is a chromatin remodeling factor involved in mediation of a plethora of signaling pathways leading to its participation in various physiological processes both during development and in adult tissues. Among other signaling pathways, the Wnt pathway has been proposed to require Brg1 for transactivation of its target genes. Given the pivotal role of the Wnt pathway in the maintenance of normal intestinal homeostasis, we aimed to investigate the effects of Brg1 loss on the intestinal physiology. To this end, we deleted Brg1 in the murine small and large intestinal epithelia using a range of transgenic approaches. Pan-epithelial loss of Brg1 in the small intestine resulted in crypt ablation, while partial Brg1 deficiency led to gradual repopulation of the intestinal mucosa with wild-type cells. In contrast, Brg1 loss in the large intestinal epithelium was compensated by upregulation of Brm. We propose that while Brg1 is dispensable for the survival and function of the progenitor and differentiated cells in the murine intestinal epithelium, it is essential for the maintenance of the stem cell population in a tissue-specific manner.

Brg1 is required for stem cell maintenance in the murine intestinal epithelium in a tissue-specific manner.

Conditional deletion of Apc in the murine intestine alters crypt-villus architecture and function. This process is accompanied by multiple changes in gene expression, including upregulation of Cited1, whose role in colorectal carcinogenesis is unknown. Here we explore the relevance of Cited1 to intestinal tumorigenesis. We crossed Cited1 null mice with Apc(Min/+) and AhCre(+)Apc(fl/fl) mice and determined the impact of Cited1 deficiency on tumour growth/initiation including tumour multiplicity, cell proliferation, apoptosis and the transcriptome. We show that Cited1 is up-regulated in both human and murine tumours, and that constitutive deficiency of Cited1 increases survival in Apc(Min/+) mice from 230.5 to 515 days. However, paradoxically, Cited1 deficiency accentuated nearly all aspects of the immediate phenotype 4 days after conditional deletion of Apc, including an increase in cell death and enhanced perturbation of differentiation, including of the stem cell compartment. Transcriptome analysis revealed multiple pathway changes, including p53, PI3K and Wnt. The activation of Wnt through Cited1 deficiency correlated with increased transcription of β-catenin and increased levels of dephosphorylated β-catenin. Hence, immediately following deletion of Apc, Cited1 normally restrains the Wnt pathway at the level of β-catenin. Thus deficiency of Cited1 leads to hyper-activation of Wnt signaling and an exaggerated Wnt phenotype including elevated cell death. Cited1 deficiency decreases intestinal tumourigenesis in Apc(Min/+) mice and impacts upon a number of oncogenic signaling pathways, including Wnt. This restraint imposed by Cited1 is consistent with a requirement for Cited1 to constrain Wnt activity to a level commensurate with optimal adenoma formation and maintenance, and provides one mechanism for tumour repression in the absence of Cited1.

Cited1 deficiency suppresses intestinal tumorigenesis.

Tumourigenesis within the intestine is potently driven by deregulation of the Wnt pathway, a process epigenetically regulated by the chromatin remodelling factor Brg1. We aimed to investigate this interdependency in an in vivo setting and assess the viability of Brg1 as a potential therapeutic target. Using a range of transgenic approaches, we deleted Brg1 in the context of Wnt-activated murine small intestinal epithelium. Pan-epithelial loss of Brg1 using VillinCreERT2 and AhCreERT transgenes attenuated expression of Wnt target genes, including a subset of stem cell-specific genes and suppressed Wnt-driven tumourigenesis improving animal survival. A similar increase in survival was observed when Wnt activation and Brg1 loss were restricted to the Lgr5 expressing intestinal stem cell population. We propose a mechanism whereby Brg1 function is required for aberrant Wnt signalling and ultimately for the maintenance of the tumour initiating cell compartment, such that loss of Brg1 in an Apc-deficient context suppresses adenoma formation. Our results highlight potential therapeutic value of targeting Brg1 and serve as a proof of concept that targeting the cells of origin of cancer may be of therapeutic relevance.

Madeleine Young

European Cancer Stem Cell Research Institute

Cardiff University

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Madeleine Young

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Cardiff University

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

European Cancer Stem Cell Research Institute