Cease and desist: modulating short-range Dpp signalling in the stem-cell niche

Drosophila ovarian germline stem cells (GSCs) are maintained by the extracellular BMP2/4 orthologue Dpp, which is produced from the surrounding somatic niche. The Dpp signal has a short range; it induces a response in GSCs within the niche, but is rapidly extinguished in their progeny only one cell-diameter away. To ensure the correct balance between stem-cell maintenance and differentiation, several regulatory mechanisms that modulate the Dpp signal at many stages of the pathway have been described. Here, we discuss the nature of the ovarian Dpp signal and review the catalogue of mechanisms that regulate it, demonstrating how the exquisite modulation of Dpp signalling in this context can result in precise and robust control of stem-cell fate. This modulation is applicable to other stem-cell environments that use BMPs as a niche signal, and the regulatory mechanisms are conceptually relevant to several other stem-cell systems.     

Integrin-dependent anchoring of a stem-cell niche

Interactions between stem cells and their surrounding microenvironment, or niche, are critical for the establishment and maintenance of stem-cell properties. The adult Drosophila testis contains a morphologically discrete stem-cell niche, the 'hub'. The small cluster of non-dividing, somatic hub cells at the anterior tip of the fly testis is contacted by the germline stem cells (GSCs), which retain their stem-cell character through the direct association with the hub. Here we show that integrin-mediated adhesion is important for maintaining the correct position of embryonic hub cells during gonad morphogenesis. The misplaced hub in integrin-deficient embryos directs the orientation of cell divisions in the presumptive GSCs, a hallmark of the active germline stem-cell niche. A decrease in integrin-mediated adhesion in adult testes, which resulted in a loss of the hub and the stem-cell population, revealed the importance of hub-cell anchoring. Finally, we show that an extracellular matrix (ECM) is present around the gonad during late embryogenesis and that this ECM is defective in integrin-deficient gonads. On the basis of our data, we propose that integrins are required for the attachment of the hub cells to the ECM, which is essential for maintaining the stem-cell niche.     

Apical meristems: the plant's fountain of youth

During postembryonic development, all organs of a plant are ultimately derived from a few pluripotent stem cells found in specialized structures called apical meristems. Here we discuss our current knowledge about the regulation of plant stem cells and their environments with main emphasis on the shoot apical meristem of Arabidopsis thaliana. Recent studies suggest that stem cells are localized in specialized niches where signals from surrounding cells maintain their undifferentiated state. In the shoot meristem, initiation of stem cells during embryogenesis, regulation of stem-cell homeostasis and termination of stem-cell maintenance during flower development appear to primarily involve regulation of the stem-cell niche.     

Stimulation of human epidermal differentiation by delta-notch signalling at the boundaries of stem-cell clusters

BACKGROUND: Human epidermis is renewed throughout life from stem cells in the basal layer of the epidermis. Signals from the surrounding keratinocytes influence the differentiation of the stem cells, but the nature of the signals is unknown. In many developing tissues, signalling mediated by the transmembrane protein Delta1 and its receptor Notch1 inhibits differentiation. Here, we investigated the role of Delta-Notch signalling in postnatal human epidermis. RESULTS: Notch1 expression was found in all living epidermal layers, but Delta1 expression was confined to the basal layer of the epidermis, with highest expression in those regions where stem cells reside. By overexpressing Delta1 or Delta(T), a truncated form of Delta1, in primary human keratinocytes and reconstituting epidermal sheets containing mixtures of Delta-overexpressing cells and wild-type cells, we found that cells expressing high levels of Delta1 or Delta(T) failed to respond to Delta signals from their neighbours. In contrast, wild-type keratinocytes that were in contact with neighbouring cells expressing Delta1 were stimulated to leave the stem-cell compartment and initiate terminal differentiation after a few rounds of division. Delta1 promoted keratinocyte cohesiveness, whereas Delta(T) did not. CONCLUSIONS: We propose that high Delta1 expression by epidermal stem cells has three effects: a protective effect on stem cells by blocking Notch signalling; enhanced cohesiveness of stem-cell clusters, which may discourage intermingling with neighbouring cells; and signalling to cells at the edges of the clusters to differentiate. Notch signalling in epidermal stem cells thus differs from other progenitor cell populations in promoting, rather than suppressing, differentiation.     

Cleavage of E-cadherin by ADAM10 mediates epithelial cell sorting downstream of EphB signalling

The formation and maintenance of complex organs requires segregation of distinct cell populations into defined territories (that is, cell sorting) and the establishment of boundaries between them. Here we have investigated the mechanism by which Eph/ephrin signalling controls the compartmentalization of cells in epithelial tissues. We show that EphB/ephrin-B signalling in epithelial cells regulates the formation of E-cadherin-based adhesions. EphB receptors interact with E-cadherin and with the metalloproteinase ADAM10 at sites of adhesion and their activation induces shedding of E-cadherin by ADAM10 at interfaces with ephrin-B1-expressing cells. This process results in asymmetric localization of E-cadherin and, as a consequence, in differences in cell affinity between EphB-positive and ephrin-B-positive cells. Furthermore, genetic inhibition of ADAM10 activity in the intestine of mice results in a lack of compartmentalization of Paneth cells within the crypt stem cell niche, a defect that phenocopies that of EphB3-null mice. These results provide important insights into the regulation of cell migration in the intestinal epithelium and may help in the understanding of the nature of the cell sorting process in other epithelial tissues where Eph-ephrin interactions play a central role.     

Environmental stimuli and intestinal stem cell behavior

Comment on: Wong VWY, et al. Nat Cell Biol 2012; 14:401-8.The intestine carries out important functions related to digestion and absorption. It is composed of three distinct layers, an outer muscle layer, a mesenchymal layer and the epithelial layer. The epithelial layer forms the protective barrier that faces the luminal content of the intestine. In order to maintain barrier function the epithelial layer needs constant replenishment. This is ensured by continuous cellular replication in proliferative crypt compartments. Following exit from the crypt, cells adopt fates along either secretory or absorptive lineage and will, after three to four days, be exfoliated into the lumen of the intestine from the tips of the villi. Intestinal stem cells located at the bottom of the proliferative crypt compartment ensure lifelong maintenance of the organ (Fig. 1A).Open in a separate windowFigure 1. Diagram of the intestinal stem cell niche. (A) Lgr5-expressing columnar-based crypt cells (CBCs) intercalated between Paneth cells are indicated in green. Stem cells located in position +4 are yellow. Lrig1 is expressed in a gradient along the niche axis with highest expression in the CBCs indicated with the thickness of the red line. Proliferation in the stem cell niche ensures continuous replenishment of the transit-amplifying (TA) compartment. (B) Within the stem cell niche, Lgr5-expressing CBCs are actively dividing and will give rise to both HopX-expressing +4 cells and TA cells. HopX-expressing cells, which are less mitotically active, will give rise to fewer TA cells and occasionally an Lgr5-expressing stem cell. Lrig1 expression in the stem cell niche reduces the amplitude of ErbB activation and is essential for controlling stem cell proliferation.Adult stem cell niches are far more heterogeneous than previously anticipated.¹ The intestinal stem cell niche can be subdivided by the relative position within the crypt. Stem cells located in position +4, just above secretory Paneth cells, express HopX, Bmi1 and Tert. These cells are generally less mitotically active than Lgr5-expressing stem cells located at the bottom of the proliferative crypts intercalated between Paneth cells (Fig. 1A).^2,3 It has been argued that both populations represent the most primitive stem cell; however, recent studies suggest that stem cells can interconvert between the two states (Fig. 1B).³ Fate mapping from cells in position 4 and at the bottom of the crypt supports this.^2,4 The positional cues responsible for cellular sorting into different functional stem cell compartments are poorly characterized. The only known effector of cellular positioning is Wnt (wingless-related MMTV integration site) signaling.⁵ Wnt is highly expressed by Paneth cells along with other mitotic factors, such as ErbB and Notch ligands.⁶ This could functionally account for the differences observed in proliferative potential along the stem cell axis. The discrete expression patterns of Lgr5 and HopX also support the existence of distinct microenvironments that supports cellular identities. A thorough characterization of the factors responsible for stem cell identity will help delineate and define the functional relationship between the distinct stem cell populations.Tissue homeostasis is governed by balanced loss and gain of cells. The stem cell niche supports constant proliferation via pro-mitotic stimuli. In order to control the amplitude of signaling strength, many pathways have developed negative feedback loops. Lrig1 (Leucine-rich repeats and immunoglobulin-like domains 1) is a negative feedback regulator of ErbB-mediated growth factor signaling.⁷ Lrig1 marks stem cells in various epithelial tissues including the intestinal epithelium, where it is expressed within the entire stem cell niche including the +4 and Lgr5-expressing cells (Fig. 1).^8,9 The functional relevance of Lrig1 and negative feedback regulation is clear from the pronounced expansion of the intestinal stem cell compartment observed in the Lrig1-KO mouse model.⁹ This is mediated via increased ErbB signaling and demonstrates the importance of balanced signaling strength within the stem cell niche.⁹ Moreover, an independent study reveals that Lrig1-KO animals have a higher incidence of colorectal cancer, suggesting that unbalanced stem cell proliferation increases tumor susceptibility.¹⁰ Future studies will address whether additional feedback regulators control signaling strength within the intestinal stem cell niche and how homeostasis within the stem cell compartment affects tumor susceptibility.     

Lrig1: a new master regulator of epithelial stem cells

Nat Cell Biol 14 4, 401–408 March042012The intestine represents the most vigorously renewing, adult epithelial tissue that makes maintenance of its homeostasis a delicate balance between proliferation, cell cycle arrest, migration, differentiation, and cell death. These processes are precisely controlled by a network of developmental signalling cascades, which include Wnt, Notch, BMP/TGFβ, and Hedgehog pathways. A new, elegant study by Wong et al (2012) now adds Lrig1 as a key player in the control of intestinal homeostasis. As for epidermal stem cells, Lrig1 limits the size of the intestinal progenitor compartment by dampening EGF/ErbB-triggered stem cell expansion.The epithelium of the small intestine is separated into two distinct compartments: a proliferative crypt, containing tissue-specific stem cells, and a villus with differentiated, short-lived cells, which are replenished by a constant stream of cell migration from the underlying crypt (Scoville et al, 2008). In particular, the canonical Wnt pathway in combination with Notch signals control stem cell maintenance and proliferation in the crypt. In addition, both pathways direct differentiation into the Paneth and the absorptive cell lineage, respectively. Intensive cross-talk between the epithelium and the underlying mesenchyme helps to define the crypt–villus boundary. This relies on epithelial-derived Hedgehog and Wnt ligands that trigger stromal BMP production, which in turn signals back to the epithelium to restrict proliferation to the crypt. A gradient of BMP antagonists produced by mesenchymal cells at the bottom of the crypts supports compartmentalization. In addition, a Wnt gradient in the crypt defines EphB expression and establishes repulsion-mediated separation into Paneth cell, proliferative, and differentiation zones along the crypt–villus axis (Figure 1A).Open in a separate windowFigure 1(A) The epithelium of the small intestine contains two populations of multipotent stem cells that reside at the bottom of the crypts. These give rise to transit-amplifying progenitors, which rapidly divide while migrating upwards. Cell cycle arrest and functional differentiation occur when these cells pass from the upper part of the crypt into the villus where they continue their upward movement until they finally undergo apoptosis. Only long-living Paneth cells follow a different path as they migrate downwards to populate the base of the crypt. Control of proliferation and lineage specification of all intestinal epithelial cells is directed in a self-organizing, dynamically regulated process based on cell–cell and cell–environment interactions. Among them, Wnt and Notch signalling have been defined as major determinants for stem cell maintenance, for proliferation of stem cells in the crypt and lineage specification. Epithelial-derived Hedgehog ligands and reciprocal stromal BMP ligands establish a connection between the epithelium and the stroma that regulates the crypt–villus boundary. In addition, repulsive interactions mediated by the Eph/ephrin family allow establishment of stable compartments. Importantly, ErbB signalling, which is partially suppressed by Lrig1 at the base of the crypt, is now shown to be a new key player in the control of stem and progenitor cell expansion. (B) Cross-talk of signalling pathways in intestinal homeostasis with an emphasis on ErbB signalling. A negative feedback loop via Lrig1 helps to fine-tune population size and proliferative activity of intestinal progenitor cells. Lrig1 has been identified as a direct target of Myc and is known to repress ErbB signalling. Myc itself is a main target of the ErbB and Wnt pathways implicated in intestinal stem and progenitor cell expansion. Moreover, Lrig1 has been found to promote BMP signalling, which interferes with intestinal proliferation by restricting AKT activation via PTEN.In the small intestine, two stem cell (SC) populations coexist: Lgr5⁺crypt base columnar cells (CBCs) that cycle every 24 h and are interspersed between Paneth cells, and slower dividing SCs concentrated above (around position +4 relative to the crypt bottom) the Lgr5⁺position (Takeda et al, 2011). The localization of these Hopx⁺mTert⁺slowly cycling SCs partly overlaps with that of quiescent cells, which show long-term label retention upon irradiation damage and pulse labelling with BrdU. Lgr5⁺CBCs are, however, dispensable (Tian et al, 2008) and can be replaced by the second stem cell population, which also shows greater activity during damage repair. The relationship between these two stem cell populations, which can reciprocally generate each other, and the mechanisms that govern quiescence are being elucidated. Importantly, leucine-rich repeats and Ig-like domains 1 (Lrig1), a transmembrane protein that interacts with ErbBs and promotes its degradation, has now been found to be enriched at the crypt base and in the progenitor compartment of the small intestine and colon (Wong et al, 2012). Lrig1 is highly expressed in Lgr5⁺, Musashi1⁺, Ascl2⁺, and Olfm4⁺CBCs, and shows an inverse relation to the pattern of activated, phosphorylated EGFR above the crypt base (Figure 1A). In line with these patterns, deletion of Lrig1 in the mouse causes a dramatic crypt expansion and increased numbers of CBCs, transit-amplifying and Paneth cells. Whether the increase of Paneth cells, which actually do not express Lrig1, is a secondary effect due to the progenitor expansion remains open. Importantly, reduction of EGFR signalling by pharmacological (Gefitinib) and genetic modulation (Egfr^wa-2 mice) is able to partially normalize all Lrig1 phenotypes. These data establish EGF/ErbB signalling, as an important regulator of the crypt compartment, and suggest Lrig1 as a central control that dampens the expansion of stem cells during normal intestinal homeostasis.Lrig1 was initially identified in the skin and proposed to maintain epidermal stem cells in a quiescent state (Watt and Jensen, 2009). Lrig1 marks human interfollicular epidermal stem cells, which can give rise to all epithelial lineages including hair follicle cells in skin reconstitution assays. However, during normal homeostasis, these cells are only bipotent, contributing to the sebaceous gland and the interfollicular epidermis. In contrast to quiescent Lrig1⁺SCs in the skin, Lrig1⁺ intestinal SCs are rapidly dividing and Lrig1 appears to only reduce their proliferative capacity. However, similar to the situation in the skin, Lrig1 and EGF signalling may play an important role during damage repair. Earlier experiments analysed the phenotype of mice lacking major EGF family members (Egger et al, 1997; Troyer et al, 2001). While these mice display some duodenal lesions during normal homeostasis, further experiments established EGF signalling as a key protective component that ameliorates mucosal damage. It remains to be seen whether activation of intestinal SCs during damage repair involves mitigation of Lrig1 dampening.Lrig1 is known to repress ErbB signalling by mediating ubiquitinylation and degradation of activated receptors, thereby limiting the amplitude of EGF signalling (Watt and Jensen, 2009). Consequently, Lrig1 deletion in the intestine induced upregulation of EGFR, ErbB2, and ErbB3, promoting downstream activation of c-Myc within intestinal stem and progenitor cells (Wong et al, 2012). Importantly, Lrig1 is a direct Myc target gene, and thereby part of a negative feedback loop that helps to fine-tune the population size and proliferative activity of intestinal progenitor cells (Figure 1B).Since the rescue of the Lrig1^−/− phenotype by EGFR deficiency was only partial (Wong et al, 2012), other mechanisms may contribute. Intriguingly, Lrig1 has been shown to promote BMP signalling by direct binding to Type I (ALK6) and Type II (ALK1, ALK2, ALK3, and ActRIB) BMP receptors (Gumienny et al, 2010). BMPR1A inactivation, deficiency of its downstream effector PTEN, and transgenic overexpression of the BMP inhibitor Noggin display crypt expansion and increased SC numbers. Inhibition of BMP signalling in these genetic models enhanced AKT activation and increased Wnt signalling, promoting proliferation and adenoma formation (Figure 1B; Scoville et al, 2008). Future work will reveal a potential involvement of BMP and Wnt signalling in the Lrig1 knockout phenotype.The ErbB pathway has been linked to inflammatory bowel disease, and progression and metastatic potential of colorectal cancer. EGFR inhibition blocks adenoma formation in preclinical models, and ErbB pathway inhibition is currently being evaluated in clinical trials with colorectal cancer patients, where promising results have been reported (Cunningham et al, 2004). In contrast, Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumours (Hedman and Henriksson, 2007). Given this heterogeneity, the Lrig1 function in tumours appears to be cell- and context-dependent. Due to early postnatal lethality of Lrig1 knockout mice, the exciting possibility that Lrig1 may act as an intestinal tumour suppressor could not be answered by the current study but clearly deserves further attention.     

Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche

Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-β. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-β-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-β.     

Computational modelling of ErbB family phosphorylation dynamics in response to transforming growth factor alpha and heregulin indicates spatial compartmentation of phosphatase activity

Members of the ErbB receptor family are associated with several cancers and appear to be providing useful targets for pharmacological therapeutics for tumours of the lung and breast. Further improvements of these therapies may be guided by a quantitative, dynamic integrative systems understanding of the complexities of ErbB dimerisation, trafficking and activation, for it is these complexities that render difficult intuiting how perturbations such as drug intervention will affect ErbB signalling activities. Towards this goal, we have developed a computational model implementing commonly accepted principles governing ErbB receptor interaction, trafficking, phosphorylation and dephosphorylation. Using this model, we are able to investigate several hypotheses regarding the compartmental localisation of dephosphorylation. Model results applied to experimental data on ErbB 1, ErbB2 and ErbB3 phosphorylation in H292 human lung carcinoma cells support a hypothesis that key dephosphorylation activity for these receptors occurs largely in an intracellular, endosomal compartment rather than at the cell surface plasma membrane. Thus, the endocytic trafficking-related compartmentalisation of dephosphorylation may define a critical aspect of the ErbB signalling response to ligand.     

Understanding hematopoietic stem-cell microenvironments

The hematopoietic system is the paradigm for adult mammalian stem-cell research. Recent advances have improved our understanding of the cellular and molecular components of the microenvironment - or niche - that regulates hematopoietic stem cells (HSCs). Here, we summarize the molecular and cellular properties of two types of niche, namely the osteoblastic and the vascular niche, in homeostatic regulation of HSC behavior, including its maintenance, proliferation, differentiation, mobilization and homing. We highlight the most recent findings and point to an important trend to the study of niche activity in cancers. Knowledge of the basic features of the HSC niches, including physical location, cell type and various signaling pathways, should provide insights into other stem-cell systems and benefit clinical applications.     

Beta1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance

The emerging evidence that stem cells develop in specialised niches highlights the potential role of environmental factors in their regulation. Here we examine the role of beta1 integrin/extracellular matrix interactions in neural stem cells. We find high levels of beta1 integrin expression in the stem-cell containing regions of the embryonic CNS, with associated expression of the laminin alpha2 chain. Expression levels of laminin alpha2 are reduced in the postnatal CNS, but a population of cells expressing high levels of beta1 remains. Using neurospheres - aggregate cultures, derived from single stem cells, that have a three-dimensional architecture that results in the localisation of the stem cell population around the edge of the sphere - we show directly that beta1 integrins are expressed at high levels on neural stem cells and can be used for their selection. MAPK, but not PI3K, signalling is required for neural stem cell maintenance, as assessed by neurosphere formation, and inhibition or genetic ablation of beta1 integrin using cre/lox technology reduces the level of MAPK activity. We conclude that integrins are therefore an important part of the signalling mechanisms that control neural stem cell behaviour in specific areas of the CNS.     

Wnt, stem cells and cancer in the intestine

The intestinal epithelium is a self-renewing tissue which represents a unique model for studying interconnected cellular processes such as proliferation, differentiation, cell migration and carcinogenesis. Although the stem cells of the intestine have not yet been physically characterized or isolated, data over the past decade have strongly implicated the Wnt/beta-catenin signalling pathway in their maintenance and progression to cancer. This review will (i) describe the distinctive features of the intestinal epithelium in relation to stem-cell function, (ii) illustrate the major genetic alterations that can lead to cancer, and (iii) show how Wnt/beta-catenin signalling controls homoeostasis in this tissue.     

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.