Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB

In the small intestine, the progeny of stem cells migrate in precise patterns. Absorptive, enteroendocrine, and goblet cells migrate toward the villus while Paneth cells occupy the bottom of the crypts. We show here that beta-catenin and TCF inversely control the expression of the EphB2/EphB3 receptors and their ligand ephrin-B1 in colorectal cancer and along the crypt-villus axis. Disruption of EphB2 and EphB3 genes reveals that their gene products restrict cell intermingling and allocate cell populations within the intestinal epithelium. In EphB2/EphB3 null mice, the proliferative and differentiated populations intermingle. In adult EphB3(-/-) mice, Paneth cells do not follow their downward migratory path, but scatter along crypt and villus. We conclude that in the intestinal epithelium beta-catenin and TCF couple proliferation and differentiation to the sorting of cell populations through the EphB/ephrin-B system.     

Modulation of stemness in a human normal intestinal epithelial crypt cell line by activation of the WNT signaling pathway

The small intestine consists of two histological compartments composed of the crypts and the villi. The function of the adult small intestinal epithelium is mediated by four different types of mature cells: enterocytes, goblet, enteroendocrine and Paneth. Undifferentiated cells reside in the crypts and produce these four types of mature cells. The niche-related Wnt and Bmp signaling pathways have been suggested to be involved in the regulation and maintenance of the stem cell microenvironment. In our laboratory, we isolated the first normal human intestinal epithelial crypt (HIEC) cell model from the human fetal intestine and in this study we investigated the expression of a panel of intestinal stem cell markers in HIEC cells under normal culture parameters as well as under conditions that mimic the stem cell microenvironment. The results showed that short term stimulation of HIEC cells with R-spondin 1 and Wnt-3a±SB-216763, a glycogen synthase kinase 3β (GSK3β) inhibitor, induced β-catenin/TCF activity and expression of the WNT target genes, cyclin D2 and LGR5. Treatment of HIEC cells with noggin, an antagonist of BMP signaling, abolished SMAD2/5/8 phosphorylation. Inducing a switch from inactive WNT/active BMP toward active WNT/inactive BMP pathways was sufficient to trigger a robust intestinal primordial stem-like cell signature with predominant LGR5, PHLDA1, PROM1, SMOC2 and OLFM4 expression. These findings demonstrate that even fully established cultures of intestinal cells can be prompted toward a CBC stem cell-like phenotype. This model should be useful for studying the regulation of human intestinal stem cell self-renewal and differentiation.     

The nature of intestinal stem cells' nurture

Mustata et al demonstrate in this issue of EMBO reports that Lgr4 expression in the stem cells and transit amplifying cells of the intestinal crypts is required for the establishment of the stem cell niche and also for the maintenance of intestinal stem cells in ex vivo organoid cultures.EMBO reports 12, 6, 558–564. doi:10.1038/embor.2011.52The ‘nature versus nurture'' debate concerns the relative contributions to an individual''s identity of its nature (that is, its genetic make-up) compared with its nurture, defined as the totality of external, environmental factors. A similar type of debate is ongoing among developmental and stem-cell biologists: is the intrinsic nature (that is, its (epi)genetic make-up) of a stem cell what makes it self-renew and differentiate according to the physiological needs of a given tissue, or is it the immediate environment (nurture) that regulates stemness? Irrespective of the relative weight of each contribution, there is little doubt that both cell-autonomous and environmental factors play crucial roles in the maintenance of homeostasis in self-renewing tissues such as the skin, mammary gland, blood and intestine. In an article published last month in EMBO reports (Mustata et al, 2011), the Lgr4 gene is shown to have a rate-limiting role in establishing the stem-cell niche of the proximal intestinal tract.…the Lgr4 gene is shown to have a rate-limiting role in establishing the stem-cell niche of the proximal intestinal tractThe epithelial lining of the proximal intestine is characterized by a unique tissue architecture consisting of villi and crypts. The intestinal crypt of Lieberkühn is a highly dynamic niche with stem cells in its lower third, which give rise to a population of fast-cycling transit-amplifying cells. Transit-amplifying cells undergo a limited number of cell divisions and eventually differentiate into four specialized cell types of the small intestine: absorptive, enteroendocrine, goblet and Paneth cells. Notably, Paneth cells are the only terminally differentiated cell type of the proximal intestinal tract that (i) move downwards along the crypt–villus axis and (ii) retain canonical Wnt signalling activity upon differentiation (van Es et al, 2005).On the basis of clonal analysis and knock-in experiments, it was shown that the crypt base columnar (CBC) cells—located in the lower third of the crypt and characterized by Lgr5 expression—represent actively cycling stem cells that are able to give rise to all differentiated cell types of the intestinal epithelium (Barker et al, 2007). More recently, it has also been shown that Paneth cells, apart from their well-known bactericidal function, are in close physical association with Lgr5⁺ stem cells, to which they provide essential niche signals such as EGF, Wnt3a and Dll4 (Sato et al, 2011). This is also important in the light of the observation that single Lgr5⁺ stem cells, when cultured ex vivo, can generate crypt–villus organoids without a (mesenchymal) niche (Sato et al, 2009). In fact, the latter is only partly true, as these organoids are cultured in matrigel and in the presence of specific growth factors that are probably released by the niche in vivo.Lgr5, together with Lgr4 and Lgr6, belongs to the family of leucine-rich repeat-containing G-protein-coupled seven-transmembrane receptors. Recently, both Lgr5 and Lgr6 have received attention from the stem-cell community: Lgr5 is a downstream Wnt target gene and a marker of cycling stem cells in the intestinal tract and the hair follicle, whereas Lgr6 expression marks adult stem cells in the skin (Barker & Clevers, 2010). However, whether they merely represent stem-cell markers or also have a functional role in stemness is unknown.Mustata et al (2011) report on the functional role of another member of the Lgr family, Lgr4, by studying the effects of a targeted loss-of-function mutation (Lgr4 KO) on the development and differentiation of the mouse small intestine both in vivo and ex vivo. Endogenous Lgr4 expression is detected in transit-amplifying cells above the Paneth-cell zone, in CBC cells, and in rare Paneth cells. Loss of Lgr4 function results in a reduction in crypt depth due to a 50% decrease in epithelial-cell proliferation and, surprisingly, in an 80% reduction in Paneth-cell differentiation. Strikingly, these phenotypic features are apparently antagonistic to those of Lgr5 KO mice, in which premature Paneth-cell development was observed (Garcia et al, 2009). Accordingly, loss of Lgr4 function partly rescues the perinatal lethality of Lgr5 KO mice indicating non-redundancy of their individual functions.Loss of Lgr4 function results in […] an 80% reduction in Paneth-cell differentiationTo further investigate the role of Lgr4 in crypt development, the ex vivo ‘minigut'' culture system (Sato et al, 2009) was used; in contrast to crypts from wild-type mice that give rise to self-renewing structures encompassing all the differentiated cell lineages of the adult gut, organoids derived from age-matched Lgr4 KO animals are initially present as hollow spheres, mainly composed of stem and transit-amplifying cells, which disaggregate within 2–3 days and die within a week in culture. In agreement with their apparently opposite and non-redundant functions, crypt cultures from Lgr5 KO mice survive long-term culture and develop into differentiated organoids comparable with those of normal mice. Whereas loss of Lgr4 function partly rescues the lethality of Lgr5 KO mice in vivo, this is not true ex vivo; compound homozygous Lgr4/5 KO crypts give rise to hollow spheres that collapse and die as observed in Lgr4 KO organoids. Hence, under these experimental conditions—that is, in the absence of a mesenchymal niche—the Lgr4 defect is dominant over the Lgr5 one.Analysis of Paneth-cell differentiation markers and of Wnt targets, including Lgr5, confirmed their downregulation in Lgr4 KO organoids, thus suggesting a role for Lgr4 in Wnt signalling. Notably, lithium chloride treatment partly rescues the ex vivo phenotype of Lgr4 KO crypts, although this is not the case for other Wnt-signalling agonists, such as Wnt3a and Gsk3β inhibitors. On the basis of these observations, the authors conclude that Lgr4 probably has a permissive, rather than a direct and active role in Wnt signalling.In view of this and other studies, a revisitation of the cell-autonomous and niche-independent features of the Lgr5⁺ cycling stem cell (CBC cells) in the intestinal crypt seems to be necessary (Fig 1). First, the capacity of CBC cells to recapitulate ex vivo the complexity of the crypt–villus unit is mostly dependent on Paneth cells (Sato et al, 2011). When they are sorted as single cells, CBC cells perform poorly in organoid formation, whereas doublets of CBC and Paneth cells show high clonogenicity (Sato et al, 2009, 2011). However, rather than occurring exclusively through the secretion of niche signals in the form of Wnt ligands, the nature of the interdependency between Paneth cells and CBC cells seems to involve additional mechanisms. As shown by Mustata et al, loss of Lgr4 function causes a Paneth-cell differentiation blockade in the presence of wild-type levels of Wnt3a and Wnt11, a defect that can be rescued by lithium chloride, but not by the Wnt3a ligand or Gsk3β inhibitors. This indicates that additional factors secreted by epithelial and possibly mesenchymal cells—for example, stromal myofibroblasts (Vermeulen et al, 2010)—and the physical association of Paneth with Lgr5⁺ cells underlies their ‘partnership'' in preserving homeostasis within such a highly dynamic tissue. Hence, Paneth cells apparently constitute an essential component of the stem-cell niche in the upper intestinal tract.…rather than occurring exclusively through the secretion of niche signals […] the nature of the interdependency between Paneth cells and CBC cells seems to involve additional mechanismsOpen in a separate windowFigure 1Schematic illustration of the intestinal stem-cell compartment in the upper intestinal tract: Lgr4 (expressed in CBC and TA cells) positively stimulates Paneth-cell differentiation and, indirectly, stem-cell homeostasis, while Lgr5 (expressed in CBC cells) has been reported to inhibit Paneth-cell differentiation (Garcia et al, 2009). CBC, crypt base columnar; Dll4, delta-like 4; EGF, epidermal growth factor; TA, transit amplifying.As it is always the case, good science leads to new questions. Which cell type provides this niche function in the colon where Paneth cells are not present? Of note, it has been shown that in the colon Lgr5⁺ cells are intermingled with yet uncharacterized CD24⁺ cells (Sato et al, 2011), a cell-surface antigen known to enrich for Paneth cells in the upper intestinal tract. As CD24 expression does not mark CBC cells, but rather their flanking cells, these observations could again reflect the supportive, niche role of Paneth cells and CD24⁺ cells in the upper and distal intestinal tract, respectively. This might also be true for colon cancer, where Paneth cells are often present, possibly to provide niche support for cancer stem cells. Alternatively, premature (in the colon) and/or fully differentiated (in the upper intestine) Paneth cells might have a dual function by providing physical and paracrine support for cycling stem cells in homeostasis, as well as representing the hitherto elusive quiescent stem cells that underlie tissue regeneration after tissue insults. Whatever the truth, the intestinal scene is now set to further dissect the complexity of the nature–nurture interaction between intestinal (cancer) stem cells and their niche.     

A genetic study of the role of the Wnt/beta-catenin signalling in Paneth cell differentiation

Wnt/β-catenin signalling plays a key role in the homeostasis of the intestinal epithelium. Whereas its role in the maintenance of the stem cell compartment has been clearly demonstrated, its role in the Paneth cell fate remains unclear. We performed genetic studies to elucidate the functions of the Wnt/β-catenin pathway in Paneth cell differentiation. We analysed mice with inducible gain-of-function mutations in the Wnt/β-catenin pathway and mice with a hypomorphic β-catenin allele that have not been previously described. We demonstrated that acute activation of Wnt/β-catenin signalling induces de novo specification of Paneth cells in both the small intestine and colon and that colon cancers resulting from Apc mutations expressed many genes involved in Paneth cell differentiation. This suggests a key role for the Wnt/β-catenin pathway in Paneth cell differentiation. We also showed that a slight decrease in β-catenin gene dosage induced a major defect in Paneth cell differentiation, but only a modest effect on crypt morphogenesis. Overall, our findings show that a high level of β-catenin activation is required to determine Paneth cell fate and that fine tuning of β-catenin signalling is critical for correct Paneth cell lineage.     

Assessment of the symmetry of stem-cell mitoses.

A model of Paneth-cell renewal in the small intestinal epithelium is used to estimate the probability that epithelial stem-cell mitoses are symmetric in the sense that they produce two cells of the same type. I found that counts of the number of Paneth cells per crypt (Paneth cells are terminally differentiated cells derived from small intestinal epithelial stem cells) support a model in which most, if not all, stem-cell mitoses are symmetric.     

Cloning of intestinal phospholipase A2 from intestinal epithelial RNA by differential display PCR

Differential display polymerase chain reaction (DD-PCR) is a powerful technique for comparing gene expression between cell types, or between stages of development or differentiation. Differentially expressed genes may be cloned and analysed further. Here we extend the use of DD-PCR to analyse differences in gene expression between two complex epithelia: that of the small intestine and of the large intestine. The aim of this study was to identify genes expressed preferentially in Paneth cells. Paneth cells are secretory epithelial cells putatively involved in host defense and regulation of crypt cell proliferation and are found at the base of the small intestinal crypts adjacent to the stem cell zone. Of 34 clones that were analysed, partial sequencing identified two clones related to known Paneth cell products: a homologue of secretory phospholipase A2 (clone B1) and a homologue of a neutrophil defensin (clone C5). B1 was strongly expressed in Paneth cells, as demonstrated by in-situ hybridization. B1 was also expressed at a lower level in the large intestinal epithelium. A full length B1 cDNA clone was isolated and sequenced, and shown to be highly homologous to type II secretory phospholipase A2 genes, and almost identical to the enhancing factor gene and the putative gene for the MOM-1 locus. B1 expression is limited to the intestinal tract, and we propose that it be designated intestinal phospholipase A2, or i -PLA2. The method we describe is well suited to the rapid identification of genes expressed exclusively or predominantly in Paneth cells.     

Modelling the spatio-temporal cell dynamics reveals novel insights on cell differentiation and proliferation in the small intestinal crypt

We developed a slow structural relaxation model to describe cellular dynamics in the crypt of the mouse small intestine. Cells are arranged in a three dimensional spiral the size of which dynamically changes according to cell production demands of adjacent villi. Cell differentiation and proliferation is regulated through Wnt and Notch signals, the strength of which depends on the local cell composition. The highest level of Wnt activity is associated with maintaining equipotent stem cells (SC), Paneth cells and common goblet-Paneth cell progenitors (CGPCPs) intermingling at the crypt bottom. Low levels of Wnt signalling area are associated with stem cells giving rise to secretory cells (CGPCPs, enteroendocrine or Tuft cells) and proliferative absorptive progenitors. Deciding between these two fates, secretory and stem/absorptive cells, depends on Notch signalling. Our model predicts that Notch signalling inhibits secretory fate if more than 50% of cells they are in contact with belong to the secretory lineage. CGPCPs under high Wnt signalling will differentiate into Paneth cells while those migrating out from the crypt bottom differentiate into goblet cells. We have assumed that mature Paneth cells migrating upwards undergo anoikis. Structural relaxation explains the localisation of Paneth cells to the crypt bottom in the absence of active forces. The predicted crypt generation time from one SC is 4-5 days with 10-12 days needed to reach a structural steady state. Our predictions are consistent with experimental observations made under altered Wnt and Notch signalling. Mutations affecting stem cells located at the crypt floor have a 50% chance of being propagated throughout the crypt while mutations in cells above are rarely propagated. The predicted recovery time of an injured crypt losing half of its cells is approximately 2 days.