Douglas J. Winton

A single type of progenitor cell maintains normal epidermis

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.

Intestinal label-retaining cells are secretory precursors expressing Lgr5

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.

Intestinal stem cell replacement follows a pattern of neutral drift.

Gut Stem Cell Replacement Gut cell turnover is characteristically rapid and relies on stem cells in the crypts that lie between the intestinal villi. The prevailing view is that stem cell division is asymmetric with one daughter retaining a stem cell character; however, this pattern of stem cell turnover does not always apply. Using long-term lineage tracing, Lopez-Garcia et al. (p. 822, published online 23 September) showed that the loss of a stem cell was compensated for by the multiplication of a neighboring cell. The rate of stem-cell loss was found to be equivalent to the rate of cell division, indicating that symmetric cell division was the rule for gut stem cells and implying stochastic expansion, contraction, and extinction of clones occurs. Intestinal stem cells form an equipotent population where loss of a stem cell is compensated for by multiplication of a neighbor. 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.

Defining Stem Cell Dynamics in Models of Intestinal Tumor Initiation

Limiting Tumor Initiation What is the competitive advantage of cells with frequently occurring mutations during tumor development? Vermeulen et al. (p. 995; see the Perspective by Bozic and Nowak) quantified the advantages of Apc-loss, Kras activation, and P53 mutation during tumor initiation in the mouse intestine. The mutations conferred only a limited clonal advantage. Indeed, many mutated stem cells were stochastically replaced by wild-type stem cells, helping to limit tumor initiation. Common genetic alterations during tumor initiation in the mouse gut reveal clonal advantages. [Also see Perspective by Bozic and Nowak] Cancer is a disease in which cells accumulate genetic aberrations that are believed to confer a clonal advantage over cells in the surrounding tissue. However, the quantitative benefit of frequently occurring mutations during tumor development remains unknown. We quantified the competitive advantage of Apc loss, Kras activation, and P53 mutations in the mouse intestine. Our findings indicate that the fate conferred by these mutations is not deterministic, and many mutated stem cells are replaced by wild-type stem cells after biased, but still stochastic events. Furthermore, P53 mutations display a condition-dependent advantage, and especially in colitis-affected intestines, clones harboring mutations in this gene are favored. Our work confirms the previously theoretical notion that the tissue architecture of the intestine suppresses the accumulation of mutated lineages.

E2f1–3 switch from activators in progenitor cells to repressors in differentiating cells

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.

Stem-cell organization in mouse small intestine

We have investigated stem-cell organization in mouse small intestine (SI) by using a cellular marker induced by somatic mutation. In small intestinal whole mounts from heterozygous Dlb-1b/ Dlb-1a mice stained with a peroxidase conjugate of Dolichos biflorus agglutinin (DBA-Px), mutations of Dlb-1bin stem cells result in loss of DBA-Px binding and so are recognizable as wholly or partly unstained crypts. The frequency of these clonal patterns can be measured during the accumulation of spontaneous mutations in untreated mice, or after treatment with ethylnitrosourea (ENU). The results show that there is a single infrequently dividing stem cell that maintains the epithelium of each crypt through a population of transit stem cells. The entire crypt epithelium is renewed approximately every 12 weeks.

Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis

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.

PPARδ status and Apc-mediated tumourigenesis in the mouse intestine

Based on recent reports that peroxisome proliferator-activated receptor delta (PPARδ) 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 β-catenin, does not cause the expected increase in PPARδ mRNA or protein but conversely, the levels of PPARδ mRNA and protein are lowered. Furthermore, we find that ApcMinPPARδ-null mice exhibit an increased predisposition to intestinal tumourigenesis. Our data suggest that PPARδ is not directly regulated by β-catenin, and that inhibition of PPARδ activity is unlikely to be an appropriate strategy for the chemoprevention or chemotherapy of intestinal malignancies.

Focal Adhesion Kinase Is Required for Intestinal Regeneration and Tumorigenesis Downstream of Wnt/c-Myc Signaling

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.

Cellular inheritance of a Cre‐activated reporter gene to determine paneth cell longevity in the murine small intestine

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 233:1332–1336, 2005.