Subtle Deregulation of the Wnt-Signaling Pathway Through Loss of Apc2 Reduces the Fitness of Intestinal Stem Cells.

The importance of the Wnt-signaling pathway on the regulation and maintenance of the intestinal stem cell (ISC) population is well recognized. However, our current knowledge base is founded on models using systems of gross deregulation of the Wnt-signaling pathway. Given the importance of this signaling pathway on intestinal homeostasis, there is a need to explore the role of more subtle alterations in Wnt-signaling levels within this tissue. Herein, we have used a model of Apc2 loss to meet this aim. Apc2 is a homolog of Apc which can also form a destruction complex capable of binding ß-catenin, albeit less efficiently than Apc. We show that systemic loss of Apc2 results in an increase in the number of cells displaying nuclear ß-catenin at the base of the intestinal crypt. This subsequently impacts the expression levels of several ISC markers and the fitness of ISCs as assessed by organoid formation efficiency. This work provides the first evidence that the function and fitness of ISCs can be altered by even minor misregulation of the Wnt-signaling pathway. Our data highlights the importance of correct maintenance of this crucial signaling pathway in the maintenance and function of the ISC population. Stem Cells 2018;36:114-122.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox 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 population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs 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. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

MBD2 deficiency does not accelerate p53 mediated lymphomagenesis.

Recent studies using hypomorphic DNA methyltransferase 1 (DNMT1) alleles have suggested that strategies aiming to reduce DNA methylation may increase genomic instability and lymphomagenesis. Given our recent finding that loss of methyl-binding domain protein 2 (Mbd2) suppresses intestinal tumorigenesis, we have tested whether loss of Mbd2 increases lymphomagenesis by intercrossing Mbd2 deficient mice with p53 deficient and p53 heterozygous mice. Unlike DNMT1, loss of Mbd2 does not accelerate lymphomagenesis, arguing that MBD2 may represent a better potential therapeutic target than DNMT1.

MBD4 deficiency reduces the apoptotic response to DNA-damaging agents in the murine small intestine.

MBD4 was originally identified through its methyl binding domain, but has more recently been characterized as a thymine DNA glycosylase that interacts with the mismatch repair (MMR) protein MLH1. In vivo, MBD4 functions to reduce the mutability of methyl-CpG sites in the genome and mice deticient in MBD4 show increased intestinal tumorigenesis on an Apc(Min/+) background. As MLH1 and other MMR proteins have been functionally linked to apoptosis, we asked whether MBD4 also plays a role in mediating the apoptotic response within the murine small intestine. Mice deficient for MBD4 showed significantly reduced apoptotic responses 6 h following treatment with a range of cytotoxic agents including gamma-irradiation, cisplatin, temozolomide and 5-fluorouracil (5-FU). This leads to increased clonogenic survival in vivo in Mbd4(-/-) mice following exposure to either 5-FU or cisplatin. We next analysed the apoptotic response to 5-FU and temozolomide in doubly mutant Mbd4(-/-), Mlh1(-/-) mice but observed no additive decrease. The results imply that MBD4 and MLH1 lie in the same pathway and therefore that MMR-dependent apoptosis is mediated through MBD4. MBD4 deficiency also reduced the normal apoptotic response to gamma-irradiation, which we show is independent of Mlh1 status (at least in the murine small intestine), so suggesting that the reliance upon MBD4 may extend beyond MMR-mediated apoptosis. Our results establish a novel functional role for MBD4 in the cellular response to DNA damage and may have implications for its role in suppressing neoplasia.